Fluorescent imaging with substituted cyanine dyes

ABSTRACT

Compounds and methods are disclosed that are useful for noninvasive imaging in the near-infrared spectral range. The cyanine compounds of Formula I are presented: 
     
       
         
         
             
             
         
       
         
         
           
             wherein 
             Q is a portion of a polymethine bridge: 
           
         
       
    
     
       
         
         
             
             
         
       
     
     Also included are bioconjugates of the compounds of Formula I, methods of labeling biomolecules with the compounds, and methods of imaging.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of PCT/US2011/057178, filed Oct. 20,2011, which application claims priority to U.S. Provisional PatentApplication Nos. 61/405,158 and 61/405,161 (both filed Oct. 20, 2010),each of which is hereby incorporated by reference for all purposes.

BACKGROUND OF THE INVENTION

Cyanine dyes have been widely used for labeling ligands or biomoleculesfor a variety of applications such as DNA sequencing. (See, for example,U.S. Pat. No. 5,571,388 for exemplary methods of identifying strands ofDNA by means of cyanine dyes). More recently, they have been used foroptical imaging of dye-labeled biomolecules, either in vivo or in vitro.(See, for example, U.S. Pat. No. 7,597,878.) Scientists favor usingcyanine dyes in biological applications because, among other reasons,many of these dyes fluoresce in the near-infrared (NIR) region of thespectrum (600-1000 nm). This makes cyanine dyes less susceptible tointerference from autofluorescence of biomolecules.

Other advantages of cyanine dyes include, for example: 1) cyanine dyesstrongly absorb and fluoresce light; 2) many cyanine dyes do not rapidlybleach under a fluorescence microscope; 3) cyanine dye derivatives canbe made that are effective coupling reagents; 4) many structures andsynthetic procedures are available, and the class of dyes is versatile;and 5) cyanine dyes are relatively small (a typical molecular weight isabout 1,000 daltons), so they do not cause appreciable stericinterference in a way that might reduce the ability of a labeledbiomolecule to reach its binding site or carry out its function.

Yet another advantage of cyanine dyes is that structural modificationscan be made by those skilled in the art that will shift the absorptionand emission curves. This is important in matching dyes to specificdetection systems and application environments. Some such modificationsmay adversely affect the performance of the dyes in other ways. Forexample, the dye fluorescence may be reduced, or the dye may stack insolution, or bind to other elements in the application system in anon-specific manner. Therefore, additional approaches for modifying thewavelength properties are of significant interest.

Despite their advantages, many of the known cyanine dyes have a numberof disadvantages. Some known cyanine dyes are not stable in the presenceof certain reagents that are commonly found in bioassays. Such reagentsinclude ammonium hydroxide, dithiothreitol (DTT), primary and secondaryamines, and ammonium persulfate (APS). Further, some known cyanine dyeslack the thermal stability and photostability that is necessary forbiological applications such as DNA sequencing, Western blotting,in-cell Western immunofluorescence assays, in vitro or in vivo opticalimaging, microscopy, and genotyping.

For these reasons, stable cyanine dyes are needed for use in labelingbiomolecules as well as in vivo imaging for the diagnosis and prognosisof diseases such as cancer. Such compositions and methods would aid inthe analysis of responses to various therapies. The present inventionsatisfies these and other needs.

BRIEF SUMMARY OF THE INVENTION

The present invention provides compounds, bioconjugates, methods oflabeling, and methods of measuring or detecting target moleculesnon-invasively, thus solving the problems of the above-described art.

As such, in one embodiment, the present invention provides a compound ofFormula I:

wherein Q is a portion of a polymethine bridge:

wherein Q is the central portion of a seven-polymethine-carbonpolymethine bridge;

each R¹ is a member selected from the group consisting of -L-Y—Z and analkyl that is additionally substituted with from 0 to 1 R¹⁴ and from 0to 1 -L-Y—Z; wherein the alkyl is optionally interrupted by at least oneheteroatom;

each R^(2a) and R^(2b) is a member independently selected from the groupconsisting of alkyl, alkenyl, hydroxyalkyl, alkoxyalkyl, aminoalkyl,amidoalkyl, alkylthioalkyl, carboxyalkyl, alkoxycarbonylalkyl, orsulfonatoalkyl; wherein a carbon of the member is additionallysubstituted with from 0 to 1 -L-Y—Z; or, alternatively, a R^(2a) andR^(2b) pair, together with the ring carbon to which the R^(2a) andR^(2b) are bonded, join to form either a spirocycloalkyl ring, whereinthe spirocycloalkyl ring is additionally substituted with from 0 to 6R¹⁴ and from 0 to 1 -L-Y—Z, or an exocyclic alkene, wherein theexocyclic alkene is additionally substituted with from 0 to 2 R¹⁴ andfrom 0 to 1 -L-Y—Z;

each R³, R⁴, R⁵, and R⁶ is a member independently selected from thegroup consisting of hydrogen, alkyl, alkenyl, halo, hydroxyl, alkoxy,amino, cyano, carboxyl, alkoxycarbonyl, amido, sulfonato, alkoxyalkyl,carboxyalkyl, alkoxycarbonylalkyl, and sulfonatoalkyl; wherein a carbonof the member is additionally substituted with from 0 to 1 -L-Y—Z; or,alternatively, a pair of said members that is selected from the groupconsisting of R³ and R⁴, R⁴ and R⁵, and R⁵ and R⁶, together with thepair of atoms to which the pair of said members is bonded, joins to forman aryl ring, wherein the aryl ring is additionally substituted withfrom 0 to 2 R¹⁴ and from 0 to 1 -L-Y—Z;

each R⁷ is a member independently selected from the group consisting ofhydrogen and alkyl; wherein the alkyl is additionally substituted withfrom 0 to 3 R¹⁴ and from 0 to 1 -L-Y—Z; or, alternatively, both R⁷,together with the intervening segment of the polyene to which both R⁷are bonded, join to form a ring, wherein said ring is selected from thegroup consisting of a cycloalkyl and a heterocyclyl ring; wherein thering is additionally substituted with from 0 to 3 R¹⁴ and from 0 to 1-L-Y—Z; and wherein the ring is optionally substituted with an exocyclicalkene, wherein the alkene is additionally substituted with from 0 to 2R¹⁴ and from 0 to 1 -L-Y—Z;

R⁸, R⁹, R¹⁰, R¹¹ and R¹² are each a member independently selected fromthe group consisting of hydrogen, alkyl, alkenyl, halo, alkoxy,sulfonato, hydroxyl, amino, carboxyl, alkoxycarbonyl, cyano, amido,thioacetyl, and -L-Y—Z; wherein at least one member selected from thegroup consisting of R⁸, R⁹, and R¹⁰ is halo;

each R¹³ is a member independently selected from the group of hydroxyl,amino, carboxyl, and alkoxycarbonyl;

each R¹⁴ is a member independently selected from the group consisting ofalkyl, alkenyl, halo, hydroxyl, alkoxy, amino, amido, amidoalkyl, cyano,cyanoalkyl, carboxyl, alkoxycarbonyl, amido, sulfonato, sulfonatoalkyl,thioacetyl, thioacetylalkyl, alkoxycarbonylalkyl, and alkoxyalkyl;wherein the alkyl or alkenyl is additionally substituted with from 0 to1 R¹³ and from 0 to 1 -L-Y—Z;

each L is an optional member independently selected from the groupconsisting of a bond, a C₁-C₂₀ alkylene, and a C₁-C₂₀ alkenylene;wherein the alkylene or alkenylene is optionally interrupted by at leastone heteroatom;

each Y is an optional member independently selected from the groupconsisting of a bond, —O—, —S—, —NH—, —NHC(O)—, —C(O)NH—, —NR¹⁵—,—NR¹⁵C(O)—, —C(O)NR¹⁵—, —N(Z)—, —N(Z)C(O)—, and —C(O)N(Z)—;

each Z is independently selected from the group consisting of -L-R¹³ and-L-R¹⁶, wherein in an alternative embodiment, —Y—Z is a member selectedfrom the group of —N(Z)₂, —N(Z)C(O)Z, and —C(O)N(Z)₂, wherein the two Zgroups may optionally be linked to form a cycloalkynyl group;

each R¹⁵ is a member independently selected from the group consisting ofalkyl and alkoxycarbonylalkyl; wherein the alkyl is optionallyinterrupted by at least one heteroatom;

each R¹⁶ is independently a member selected from the group consisting ofactivated acyl, acrylamido, optionally substituted alkylsulfonate ester,azido, optionally substituted arylsulfonate ester, optionallysubstituted amino, aziridino, boronato, cycloalkynyl,cycloalkynylcarbonyl, diazo, formyl, glycidyl, halo, haloacetamidyl,haloalkyl, haloplatinato, halotriazino, hydrazinyl, imido ester,isocyanato, isothiocyanato, maleimidyl, mercapto, phosphoramidityl, aphotoactivatable moiety, vinyl sulfonyl, alkynyl, cycloalkynyl,spirocycloalkynyl, a pegylated azido, a pegylated alkynyl, a pegylatedcycloalkynyl, a pegylated spirocycloalkynyl, an o-diarylphosphino arylester, and an ortho substituted phosphine oxide aryl ester;

and wherein said compound has a balanced charge.

Preferably, said compound has at least one -L-Y—Z group; morepreferably, said compound has exactly one -L-Y—Z group.

In another embodiment, the present invention provides a bioconjugate ofthe Formula II:

wherein Q^(L) is a portion of a polymethine bridge:

wherein Q^(L) is the central portion of a seven-polymethine-carbonpolymethine bridge;

wherein R¹, R^(2a), R^(2b), R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹²,R¹³, R¹⁴, R¹⁵, L, and Y are as previously defined for the compound ofFormula I;

each Z is independently selected from the group consisting of -L-R¹³ and-L-R^(L);

each R^(L) comprises a linking group and a biomolecule connectedthereby, wherein the compound comprises at least one R^(L), and whereinthe compound has a balanced charge. Preferably, the compound comprisesexactly one R^(L).

In yet another embodiment, the present invention provides a method orprocess for labeling a ligand or biomolecule with a compound of FormulaI, the method comprising contacting a ligand or biomolecule with acompound having Formula I to generate the corresponding bioconjugatecompound of Formula II.

In still yet another embodiment, the compounds of Formula I or II can beused as in vitro or in vivo optical imaging agents of tissues and organsin various biomedical applications. In one aspect, the present inventionprovides a method for imaging, the method comprising administering acompound of Formula I or Formula II.

Further aspects, objects, and advantages of the invention will becomeapparent upon consideration of the detailed description and figures thatfollow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the absorbance and emission spectra of compound 11 inphosphate-buffered saline (PBS). For emission, the y-axis indicates thedye's fluorescence as expressed in arbitrary units.

FIG. 2 shows the absorbance and emission spectra of compound 16 (Example16) in PBS. For emission, the y-axis indicates the dye's fluorescence asexpressed in arbitrary units.

FIG. 3 shows the signal intensity of a goat anti-mouse (GAM) conjugatewith compound 17 at different dye/protein (D/P) ratios and 0.2 μg/mLdilution. IRDye® 800CW was used as a control.

FIG. 4 shows the signal intensity of a goat anti-mouse (GAM) conjugatewith compound 17 at different dye/protein (D/P) ratios and 0.2 ng/mLdilution. IRDye® 800CW was used as a control.

FIG. 5 illustrates a Western blot of a goat anti-mouse (GAM) conjugatewith compound 17 (0.2 μg/mL). IRDye® 800CW was used as a control.

FIG. 6 illustrates a Western blot of a goat anti-mouse (GAM) conjugatewith compound 17 (0.2 ng/mL). IRDye® 800CW was used as a control.

FIG. 7 illustrates an immunocytochemical assay showing U87GM binding a17/RGD conjugate (A), a U87GM challenge with unlabeled RGD (B), andU87GM evaluation of 16 for a non-specific dye effect (C), animmunocytochemical assay showing PC3MLN4 binding a 17/RGD conjugate (D),a PC3MLN4 challenge with unlabeled RGD (E), and PC3MLN4 evaluation of 16for a non-specific dye effect (F).

FIG. 8 A) Binding results in U87GM cells with either a 17/RGD conjugateor IRDye® 800CW/RGD. B) Blocking assay in U87GM cells: 17/RGD conjugate(200 nM) addition with a serial dilution of unlabeled RGD (0.06-30 μM).

FIG. 9 Compound 16 and IRDye 800CW carboxylate clearance series showsvery little difference between the two dyes, indicating good clearancein vivo: Dorsal (A) or ventral (B) views. A graphical representation ofthe whole-body core signal from the dorsal view is presented (C).

DETAILED DESCRIPTION OF THE INVENTION Definitions

The terms “a,” “an,” or “the” as used herein not only include aspectswith one member, but also include aspects with more than one member. Forexample, an embodiment of a method of imaging that comprises using acompound set forth in claim 1 would include an aspect in which themethod comprises using two or more compounds set forth in claim 1.

“The term “about” as used herein to modify a numerical value indicates aclose range around that explicit value. If “X” were the value, “about X”would indicate a value from 0.9X to 1.1X, and more preferably, a valuefrom 0.95X to 1.05X. Any reference to “about X” specifically indicatesat least the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X,1.03X, 1.04X, and 1.05X. Thus, “about X” is intended to teach andprovide written description support for a claim limitation of, e.g.,“0.98X.” When the quantity “X” only allows whole-integer values (e.g.,“X carbons”) and X is at most 15, “about X” indicates from (X−1) to(X+1). In this case, “about X” as used herein specifically indicates atleast the values X, X−1, and X+1. If X is at least 16, the values of0.90X and 1.10X are rounded to the nearest whole-integer values todefine the boundaries of the range.

When the modifier “about” is applied to describe the beginning of anumerical range, it applies to both ends of the range. Thus, “from about700 to 850 nm” is equivalent to “from about 700 nm to about 850 nm.”When “about” is applied to describe the first value of a set of values,it applies to all values in that set. Thus, “about 680, 700, or 750 nm”is equivalent to “about 680 nm, about 700 nm, or about 750 nm.” However,when the modifier “about” is applied to describe only the end of therange or only a later value in the set of values, it applies only tothat value or that end of the range. Thus, the range “about 2 to 10” isthe same as “about 2 to about 10,” but is different from the range “2 toabout 10.”

“Activated acyl” as used herein includes a —C(O)-LG group. “Leavinggroup” or “LG” is a group that is susceptible to displacement by anucleophilic acyl substitution (i.e., a nucleophilic addition to thecarbonyl of —C(O)-LG, followed by elimination of the leaving group).Representative leaving groups include halo, cyano, azido, carboxylicacid derivatives such as t-butylcarboxy, and carbonate derivatives suchas i-BuOC(O)O—. An activated acyl group may also be an activated esteras defined herein or a carboxylic acid activated by a carbodiimide toform an anhydride or mixed anhydride —OC(O)R^(a) or —OC(NR^(a))NHR^(b),wherein R^(a) and R^(b) are members independently selected from thegroup consisting of C₁-C₆ alkyl, C₁-C₆ perfluoroalkyl, C₁-C₆ alkoxy,cyclohexyl, 3-dimethylaminopropyl, or N-morpholinoethyl. Preferredactivated acyl groups include activated esters.

“Activated ester” as used herein includes a derivative of a carboxylgroup that is more susceptible to displacement by nucleophilic additionand elimination than an ethyl ester group (e.g., an NHS ester, asulfo-NHS ester, a PAM ester, or a halophenyl ester). Representativecarbonyl substituents of activated esters include succinimidyloxy(—OC₄H₄NO₂), sulfosuccinimidyloxy (—OC₄H₃NO₂SO₃H),-1-oxybenzotriazolyl(—OC₆H₄N₃); 4-sulfo-2,3,5,6-tetrafluorophenyl; or anaryloxy group that is optionally substituted one or more times byelectron-withdrawing substituents such as nitro, fluoro, chloro, cyano,trifluoromethyl, or combinations thereof (e.g., pentafluorophenyloxy).Preferred activated esters include succinimidyloxy andsulfosuccinimidyloxy esters.

“Acyl” as used herein includes an alkanoyl, aroyl, heterocycloyl, orheteroaroyl group as defined herein. Representative acyl groups includeacetyl, benzoyl, nicotinoyl, and the like.

“Alkanoyl” as used herein includes an alkyl-C(O)— group wherein thealkyl group is as defined herein. Representative alkanoyl groups includeacetyl, ethanoyl, and the like.

“Alkenyl” as used herein includes a straight or branched aliphatichydrocarbon group of 2 to about 15 carbon atoms that contains at leastone carbon-carbon double bond. Preferred alkenyl groups have 2 to about12 carbon atoms. More preferred alkenyl groups contain 2 to about 6carbon atoms. “Lower alkenyl” as used herein includes alkenyl of 2 toabout 6 carbon atoms. Representative alkenyl groups include vinyl,allyl, n-butenyl, 2-butenyl, 3-methylbutenyl, n-pentenyl, heptenyl,octenyl, decenyl, and the like.

“Alkenylene” as used herein includes a straight or branched bivalenthydrocarbon chain containing at least one carbon-carbon double or triplebond. Preferred alkenylene groups include from 2 to about 12 carbons inthe chain, and more preferred alkenylene groups include from 2 to 6carbons in the chain. In one aspect, hydrocarbon groups that contain acarbon-carbon double bond are preferred. In a second aspect, hydrocarbongroups that contain a carbon-carbon triple bond are preferred.Representative alkenylene groups include —CH═CH—, —CH₂—CH═CH—,—C(CH₃)═CH—, —CH₂CH═CHCH₂—, ethynylene, propynylene, n-butynylene, andthe like.

“Alkoxy” as used herein includes an alkyl-O— group wherein the alkylgroup is as defined herein. Representative alkoxy groups includemethoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, heptoxy, and the like.

“Alkoxyalkyl” as used herein includes an alkyl-O-alkylene-group whereinalkyl and alkylene are as defined herein. Representative alkoxyalkylgroups include methoxyethyl, ethoxymethyl, n-butoxymethyl andcyclopentylmethyloxyethyl.

“Alkoxycarbonyl” as used herein includes an ester group; i.e., analkyl-O—CO— group wherein alkyl is as defined herein. Representativealkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl,t-butyloxycarbonyl, and the like.

“Alkoxycarbonylalkyl” as used herein includes analkyl-O—CO-alkylene-group wherein alkyl and alkylene are as definedherein. Representative alkoxycarbonylalkyl includemethoxycarbonylmethyl, ethoxycarbonylmethyl, methoxycarbonylethyl, andthe like.

“Alkyl” as used herein includes an aliphatic hydrocarbon group, whichmay be straight or branched-chain, having about 1 to about 20 carbonatoms in the chain. Preferred alkyl groups have 1 to about 12 carbonatoms in the chain. More preferred alkyl groups have 1 to 10 or 1 to 6carbon atoms in the chain. “Branched-chain” as used herein includes thatone or more lower alkyl groups such as methyl, ethyl or propyl areattached to a linear alkyl chain. “Lower alkyl” as used herein includes1 to about 6 carbon atoms, preferably 5 or 6 carbon atoms in the chain,which may be straight or branched. Representative alkyl groups includemethyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, and3-pentyl.

“Alkylene” as used herein includes a straight or branched bivalenthydrocarbon chain of 1 to about 6 carbon atoms. Preferred alkylenegroups are the lower alkylene groups having 1 to about 4 carbon atoms.Representative alkylene groups include methylene, ethylene, and thelike.

“Alkylsulfonate ester” as used herein includes an alkyl-SO₃— groupwherein the alkyl group is as defined herein. Preferred alkylsulfonateester groups are those wherein the alkyl group is lower alkyl.Representative alkylsulfonate ester groups include mesylate ester (i.e.,methylsulfonate ester).

An “optionally substituted” alkylsulfonate ester includes analkylsulfonate ester as defined herein, wherein the aryl group isadditionally substituted with from 0 to 3 halo, alkyl, aryl, haloalkyl,or haloaryl groups as defined herein. Preferred optionally substitutedalkylsulfonate groups include triflate ester (i.e.,trifluoromethylsulfonate ester).

“Alkylthio” as used herein includes an alkyl-S— group wherein the alkylgroup is as defined herein. Preferred alkylthio groups are those whereinthe alkyl group is lower alkyl. Representative alkylthio groups includemethylthio, ethylthio, isopropylthio, heptylthio, and the like.

“Alkylthioalkyl” as used herein includes an alkylthio-alkylene-groupwherein alkylthio and alkylene are defined herein. Representativealkylthioalkyl groups include methylthiomethyl, ethylthiopropyl,isopropylthioethyl, and the like.

“Alkynyl” as used herein includes a straight or branched aliphatichydrocarbon group of 2 to about 15 carbon atoms that contains at leastone carbon-carbon triple bond. Preferred alkynyl groups have 2 to about12 carbon atoms. More preferred alkynyl groups contain 2 to about 6carbon atoms. “Lower alkynyl” as used herein includes alkynyl of 2 toabout 6 carbon atoms. Representative alkynyl groups include propynyl,2-butynyl, 3-methylbutynyl, n-pentynyl, heptynyl, and the like.

“Amido” as used herein includes a group of formula Y₁Y₂N—C(O)— whereinY₁ and Y₂ are independently hydrogen, alkyl, or alkenyl; or Y₁ and Y₂,together with the nitrogen through which Y₁ and Y₂ are linked, join toform a 4- to 7-membered azaheterocyclyl group (e.g., piperidinyl).Representative amido groups include primary amido (H₂N—C(O)—),methylamido, dimethylamido, diethylamido, and the like. Preferably,“amido” is an —C(O)NRR′ group where R and R′ are members independentlyselected from the group consisting of H and alkyl. More preferably, atleast one of R and R′ is H.

“Amidoalkyl” as used herein includes an amido-alkylene-group whereinamido and alkylene are defined herein. Representative amidoalkyl groupsinclude amidomethyl, amidoethyl, dimethylamidomethyl, and the like.

“Amino” as used herein includes a group of formula Y₁Y₂N— wherein Y₁ andY₂ are independently hydrogen, acyl, aryl, or alkyl; or Y₁ and Y₂,together with the nitrogen through which Y₁ and Y₂ are linked, join toform a 4- to 7-membered azaheterocyclyl group (e.g., piperidinyl).Optionally, when Y₁ and Y₂ are independently hydrogen or alkyl, anadditional substituent can be added to the nitrogen, making a quaternaryammonium ion. Representative amino groups include primary amino (H₂N—),methylamino, dimethylamino, diethylamino, tritylamino, and the like.Preferably, “amino” is an —NRR′ group where R and R′ are membersindependently selected from the group consisting of H and alkyl.Preferably, at least one of R and R′ is H.

“Aminoalkyl” as used herein includes an amino-alkylene-group whereinamino and alkylene are defined herein. Representative aminoalkyl groupsinclude aminomethyl, aminoethyl, dimethylaminomethyl, and the like.

“Aroyl” as used herein includes an aryl-CO— group wherein aryl isdefined herein. Representative aroyl include benzoyl, naphth-1-oyl andnaphth-2-oyl.

“Aryl” as used herein includes an aromatic monocyclic or multicyclicring system of 6 to about 14 carbon atoms, preferably of 6 to about 10carbon atoms. Representative aryl groups include phenyl and naphthyl.

“Arylsulfonate ester” as used herein includes an aryl-SO₃ group whereinthe aryl group is as defined herein. Representative arylsulfonate estergroups include phenylsulfonate ester.

An “optionally substituted” arylsulfonate ester includes anarylsulfonate ester as defined herein, wherein the aryl group isadditionally substituted with from 0 to 3 halo, alkyl, aryl, haloalkyl,or haloaryl groups as defined herein. Preferred optionally substitutedarylsulfonate esters include tosylate ester (i.e., p-tolylsulfonateester).

“Aromatic ring” as used herein includes 5-12 membered aromaticmonocyclic or fused polycyclic moieties that may include from zero tofour heteroatoms selected from the group consisting of oxygen, sulfur,selenium, and nitrogen. Exemplary aromatic rings include benzene,pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole,pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, naphthalene,benzathiazoline, benzothiophene, benzofurans, indole, benzindole,quinoline, and the like. The aromatic ring group can be substituted atone or more positions with halo, alkyl, alkoxy, alkoxy carbonyl,haloalkyl, cyano, sulfonato, amino sulfonyl, aryl, sulfonyl,aminocarbonyl, carboxy, acylamino, alkyl sulfonyl, amino and substitutedor unsubstituted substituents.

“Balanced charge” as used herein includes the condition that the netcharge for a compound and its associated counterions be zero understandard physiological conditions. In order to achieve a balancedcharge, a skilled person will understand that after the first additionalsulfonato group that balances the +1 charge of the indolinium ring ofthe compounds herein, a cationic counterion (e.g., the cation of a GroupI metal such as sodium) must be added to balance the negative chargefrom additional sulfonato groups. Similarly, anionic counterions must beadded to balance any additional cationic groups (e.g., most amino groupsunder physiological conditions).

“Biomolecule” as used herein includes a natural or synthetic moleculefor use in biological systems. Preferred biomolecules include a protein,a peptide, an enzyme substrate, a hormone, an antibody, an antigen, ahapten, an avidin, a streptavidin, a carbohydrate, a carbohydratederivative, an oligosaccharide, a polysaccharide, a nucleic acid, adeoxynucleic acid, a fragment of DNA, a fragment of RNA, nucleotidetriphosphates, acyclo terminator triphosphates, PNA, and the like. Morepreferred biomolecules include a protein, a peptide, an antibody, anavidin, a streptavidin, and the like. Even more preferred biomoleculesinclude a peptide, an antibody, an avidin, and a streptavidin.

“Carboxy” and “carboxyl” as used herein include a HOC(O)— group (i.e., acarboxylic acid) or a salt thereof.

“Carboxyalkyl” as used herein includes a HOC(O)-alkylene-group whereinalkylene is defined herein. Representative carboxyalkyls includecarboxymethyl (i.e., HOC(O)CH₂—) and carboxyethyl (i.e., HOC(O)CH₂CH₂—).

“Cycloalkenyl” as used herein includes a cyclic hydrocarbon group of 4to about 15 carbon atoms that contains at least one carbon-carbondouble. The cycloalkenyl ring may include from 0 to 6 R¹⁴ substituentsand 0 to 2 R^(L) substituents, and when present, the ring-fused aryl orheteroaryl rings may also include from 0 to 4 R¹⁴ substituents and 0 to2 R^(L) substituents. The R¹⁴ and R^(L) substituents are as otherwisedefined herein. Preferred alkenyl groups have 5 to about 12 carbonatoms. More preferred alkenyl groups contain 7 to about 14 carbon atoms.Representative cycloalkenyl groups include cyclopentenyl, cyclohexenyl,

“Cycloalkynyl” as used herein includes a cyclic hydrocarbon group of 5to about 15 carbon atoms that contains at least one carbon-carbon triplebond. In a preferred aspect, the cyclic hydrocarbon may optionally beinterrupted by a heteroatom (e.g., N, O, S; preferably N) and mayinclude at least one ring-fused aryl or heteroaryl ring (e.g., DBCO orDBCO-1). The cycloalkynyl ring may include from 0 to 6 R¹⁴ substituentsand 0 to 2 R^(L) substituents, and when present, the ring-fused aryl orheteroaryl rings may also include from 0 to 4 R¹⁴ substituents and 0 to2 R^(L) substituents. The R¹⁴ and R^(L) substituents are as otherwisedefined herein. In some aspect, the R^(L) substituent includes aring-fused heteroaryl group as part of the linking group with thebiomolecule (e.g., the reaction of DBCO with an azide-substitutedbiomolecule). Preferred alkynyl groups have 5 to about 12 carbon atoms.More preferred alkynyl groups contain 7 to about 14 carbon atoms.Representative cycloalkynyl groups include cyclopentynyl, cyclohexynyl,cyclooctynyl, dibenzocyclooctynyl (or DBCO, which includes a nitrogen inthe “octyne” ring or DBCO-1), BARAC, DIFO, DIBO, TMDIBO, DIFO3 and thelike.

“Cycloalkynylcarbonyl” includes the definition of cycloalkynyl abovewith an exocylic carbonyl, for example, a dibenzocyclooctynylcarbonyl orC(O)DBCO, which includes a nitrogen in the “octyne” ring and anexocyclic carbonyl group, and the like.

“Cycloalkyl” as used herein includes a non-aromatic mono- or multicyclicring system of about 3 to about 10 carbon atoms, preferably of about 5to about 10 carbon atoms. More preferred cycloalkyl rings contain 5 or 6ring atoms. A cycloalkyl group optionally comprises at least onesp²-hybridized carbon (e.g., a ring incorporating an endocyclic orexocyclic olefin). Representative monocyclic cycloalkyl groups includecyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, and the like.Representative multicyclic cycloalkyl include 1-decalin, norbornyl,adamantyl, and the like.

“Cycloalkylene” as used herein includes a bivalent cycloalkyl havingabout 4 to about 8 carbon atoms. Preferred cycloalkylenyl groups include1,2-, 1,3-, or 1,4-cis- or trans-cyclohexylene.

“Cyanine dye” as used herein includes a compound having two substitutedor unsubstituted nitrogen-containing heterocyclic rings joined by anunsaturated bridge. Examples include the structures of Formula I.

“Exocyclic alkene” or “exocyclic olefin” as used interchangeably hereinincludes an alkene having one alkene carbon that is part of a ring andthe other alkene carbon not part of the same ring, though it may beincluded within a second ring. The second alkene carbon can beunsubstituted or substituted. If the second alkene carbon isdisubstituted, the substituents can be the same (e.g., 1,1-dimethylsubstitution) or different (e.g., 1-methyl-1-(2-ethoxyethyl)substitution). Examples of compounds with exocyclic alkenes includemethylenecyclohexane; (E)-1-ethylidene-2,3-dihydro-1H-indene;pentan-3-ylidenecycloheptane; 2-cyclobutylidenepropan-1-ol; and(3-methoxycyclopent-2-enylidene)cyclohexane.

“Geminal” substituents as used herein include two or more substituentsthat are directly attached to the same atom. An example is 3,3-dimethylsubstitution on a cyclohexyl or spirocyclohexyl ring.

“Halo” or “halogen” as used herein includes fluoro, chloro, bromo, oriodo.

“Haloalkyl” as used herein includes an alkyl group wherein the alkylgroup includes one or more halo-substituents.

“Haloaryl” as used herein includes an alkyl group wherein the aryl groupincludes one or more halo-substituents.

“Heptamethine” as used herein includes a polymethine containing sevenpolymethine carbons. In a preferred embodiment, the heptamethine issubstituted at the 4-position.

“Heteroatom” as used herein includes an atom other than carbon orhydrogen. Representative heteroatoms include O, S, and N. The nitrogenor sulphur atom of the heteroatom is optionally oxidized to thecorresponding N-oxide, S-oxide (sulfoxide), or S,S-dioxide (sulfone). Ina preferred aspect, a heteroatom has at least two bonds to alkylenecarbon atoms (e.g., —C₁-C₉ alkylene-O—C₁-C₉ alkylene-). In someembodiments, a heteroatom is further substituted with an acyl, alkyl,aryl, cycloalkyl, heterocyclyl, or heteroaryl group (e.g., —N(Me)—;—N(Ac)—).

“Heteroaroyl” as used herein includes a heteroaryl-C(O)— group whereinheteroaryl is as defined herein. Representative heteroaroyl groupsinclude thiophenoyl, nicotinoyl, pyrrol-2-ylcarbonyl, pyridinoyl, andthe like.

“Heterocycloyl” as used herein includes a heterocyclyl-C(O)— groupwherein heterocyclyl is as defined herein. Representative heterocycloylgroups include N-methyl prolinoyl, tetrahydrofuranoyl, and the like.

“Heterocyclyl” as used herein includes a non-aromatic saturatedmonocyclic or multicyclic ring system of about 3 to about 10 ring atoms,preferably about 5 to about 10 ring atoms, in which one or more of theatoms in the ring system is an element or elements other than carbon,e.g., nitrogen, oxygen or sulfur. Preferred heterocyclyl groups containabout 5 to about 6 ring atoms. A heterocyclyl group optionally comprisesat least one sp²-hybridized atom (e.g., a ring incorporating a carbonyl,endocyclic olefin, or exocyclic olefin). The prefix “aza,” “oxa,” or“thia” before heterocyclyl means that at least a nitrogen, oxygen orsulfur atom respectively is present as a ring atom. The nitrogen orsulphur atom of the heterocyclyl is optionally oxidized to thecorresponding N-oxide, S-oxide or S,S-dioxide. Representative monocyclicheterocyclyl rings include piperidyl, pyrrolidinyl, piperazinyl,morpholinyl, thiomorpholinyl, thiazolidinyl, 1,3-dioxolanyl,1,4-dioxanyl, tetrahydrofuranyl, tetrahydrothiophenyl,tetrahydrothiopyranyl, and the like.

“Heterocyclylene” as used herein includes a bivalent heterocyclyl group.Representative cycloalkylenyl groups include 1,2-, 1,3-, or1,4-piperidinylene as well as 2,3- or 2,4-cis- or trans-piperidinylene.

“Heteroaryl” as used herein includes an aromatic monocyclic ormulticyclic ring system of about 5 to about 14 ring atoms, preferablyabout 5 to about 10 ring atoms, in which at least one of the atoms inthe ring system is an element other than carbon, i.e., nitrogen, oxygenor sulfur. Preferred heteroaryls contain about 5 to about 6 ring atoms.The prefix “aza,” “oxa,” or “thia” before heteroaryl means that at leasta nitrogen, oxygen or sulfur atom respectively is present as a ringatom. A nitrogen atom of a heteroaryl is optionally oxidized to thecorresponding N-oxide. Representative heteroaryls include pyrazinyl,furanyl, thienyl, pyridyl, pyrimidinyl, isoxazolyl, isothiazolyl,oxazolyl, thiazolyl, pyrazolyl, furazanyl, pyrrolyl, pyrazolyl,triazolyl, 1,2,4-thiadiazolyl, pyrazinyl, pyridazinyl, quinoxalinyl,phthalazinyl, imidazo[1,2-a]pyridine, imidazo[2,1-b]thiazolyl,benzofurazanyl, indolyl, azaindolyl, benzimidazolyl, benzothienyl,quinolinyl, imidazolyl, thienopyridyl, quinazolinyl, thienopyrimidyl,pyrrolopyridyl, imidazopyridyl, isoquinolinyl, benzoazaindolyl,1,2,4-triazinyl, benzothiazolyl and the like.

“Hydroxyalkyl” as used herein includes an alkyl group as defined hereinsubstituted with one or more hydroxy groups. Preferred hydroxyalkylscontain lower alkyl. Representative hydroxyalkyl groups includehydroxymethyl and 2-hydroxyethyl.

When any two substituent groups or any two instances of the samesubstituent group are “independently selected” from a list ofalternatives, they may be the same or different. For example, if R^(a)and R^(b) are independently selected from the group consisting ofmethyl, hydroxymethyl, ethyl, hydroxyethyl, and propyl, then a moleculewith two R^(a) groups and two R^(b) groups could have all groups bemethyl. Alternatively, the first R^(a) could be methyl, the second R^(a)could be ethyl, the first R^(b) could be propyl, and the second R^(b)could be hydroxymethyl (or any other substituents taken from the group).Alternatively, both R^(a) and the first R^(b) could be ethyl, while thesecond R^(b) could be hydroxymethyl (i.e., some pairs of substituentgroups may be the same, while other pairs may be different).

“Linking group” as used herein includes the atoms joining a compound ofFormula I with a biomolecule. Table 1 includes a list of preferred bondsfor linking groups (i.e., Column C); the linking group comprises theresulting bond and optionally can include additional atoms. See also R.Haugland, Molecular Probes Handbook of Fluorescent Probes and ResearchChemicals, Molecular Probes, Inc. (1992). In one embodiment, R¹⁶represents a linking group precursor before the attachment reaction witha biomolecule, and R^(L) represents the resultant attachment between thecompound of Formula I and the biomolecule (i.e., R^(L) comprises thelinking group and the biomolecule linked thereby). Preferred reactivefunctionalities include phosphoramidite groups, an activated ester(e.g., an NHS ester), thiocyanate, isothiocyanate, maleimide andiodoacetamide.

“Methine carbon” or “polymethine carbon” as used herein includes acarbon that is directly connecting the two heterocyclic rings by meansof the polymethine bridge. In a preferred embodiment, at least onepolymethine carbon of a polymethine bridge is additionally substitutedwith another group such as alkyl, cycloalkyl, or aryl (e.g.,—CH═CH—C(Ar)═CH—CH═ or ═CH—CH═C(Ar)—(CH═CH)₂—).

“Oxo” as used herein includes a group of formula >C═O (i.e., a carbonylgroup —C(O)—).

“Pentamethine” as used herein includes a polymethine containing fivepolymethine carbons. In a preferred embodiment, the pentamethine issubstituted at the 3-position.

“A “photoactivatable moiety” as used herein includes a chemical group ormolecule that, upon exposure to light, absorbs a photon to enter anexcited state. The excited-state group or molecule undergoes a chemicalreaction or series of reactions. Alternatively, the excitation changesthe light-emitting properties of the group or molecules (e.g.,photoactivatable fluorescent dyes). Examples of photoactivatablemoieties include aryl azides, benzophenones (e.g., 4-benzoyloxybenzoicacid as well as its esters and amides), nitroaryl groups (e.g.,5-carboxymethoxy-2-nitrobenzyl (CMNB); α-carboxy-2-nitrobenzyl (CNB);4,5-dimethoxy-2-nitrobenzyl (DMNB); 1-(4,5-dimethoxy-2-nitrophenyl)ethyl(DMNPE); nitrophenyl (NP); and 1-(2-nitrophenyl)ethyl (NPE) groups),coumarins, diazo groups, photoactivatable fluorescent dyes (e.g.,5-carboxyfluorescein-bis-(5-carboxymethoxy-2-nitrobenzyl)ether,β-alanine-carboxamide, succinimidyl ester), and tetrazoles.

“Polyene” as used herein includes a straight or branched bivalenthydrocarbon chain containing at least two “alkenylene” groups as definedherein that are in conjugation. The polyene is optionally substitutedwith one or more “alkylene group substituents” as defined herein. Aportion of the polyene may be incorporated into a ring (i.e., ═C(R)—,

wherein R and the terminal bond are linked in a larger ring; or—C(R¹)═C(R²)—, wherein R¹ and R² are linked in a larger ring).Representative polyenes include —CH═CH—CH═CH—, —CH═CH—C(Ar)═CH—CH═C(R)—,—C(R)═CH—CH═C(Ar)-(CH═CH)₂—, and the like.

“Polymethine” or “polymethine bridge” as used herein includes the seriesof conjugated, sp²-hybridized carbons that form the unsaturated bridgedirectly connecting the two nitrogen-containing heterocyclic rings of acompound of Formula I. In a preferred embodiment, the polymethine hasfive or seven carbons directly connecting the heterocyclic rings (i.e.,pentamethine or heptamethine).

“Phosphoramidityl” as used herein includes a trivalent phosphorous atombonded to two alkoxy groups and an amino group.

“Spirocycloalkyl” as used herein includes a cycloalkyl in which geminalsubstituents on a carbon atom are replaced to form a 1,1-substitutedring.

“Sulfonato” as used herein includes an —SO₃ ⁻ group, preferably balancedby a cation such as H⁺, Na⁺, K⁺, and the like.

“Sulfonatoalkyl” as used herein includes a sulfonato-alkylene-groupwherein sulfonato and alkylene are as defined herein. A more preferredembodiment includes alkylene groups having from 2 to 6 carbon atoms, anda most preferred embodiment includes alkylene groups having 2, 3, or 4carbons. Representative sulfonatoalkyls include sulfonatomethyl,3-sulfonatopropyl, 4-sulfonatobutyl, 5-sulfonatopentyl,6-sulfonatohexyl, and the like.

In general, the unit prefix “u” as used herein is equivalent to “g” or“micro.” For example, “ul” is equivalent to “μl” or “microliters.”

Cyanine Dye Compounds

In one embodiment, the present invention provides a compound of FormulaI:

wherein Q is a three-methine-carbon segment:

wherein the segment is the central portion of a seven-methine-carbonpolymethine bridge.

In a preferred aspect, Q is a portion of a polymethine bridge that is apentamethine:

In a second preferred aspect, Q is a portion of a polymethine bridgethat is a heptamethine:

In an alternative preferred aspect, the polymethine bridge is asubstituted heptamethine:

More preferably, the substituted heptamethine includes a cycloalkylring:

In a preferred aspect, a dye is symmetric. Non-symmetric dyes are oftenmore difficult to synthesize in high purity and yield. (See U.S. Pat.No. 6,747,159 for some advantages of symmetric dyes). However, as setforth in the Examples, some embodiments of the present invention arenon-symmetric dyes.

Each R¹ is an independently selected alkyl group that is additionallysubstituted with from 0 to 1 R¹⁴ and from 0 to 1 -L-Y—Z; wherein thealkyl is optionally interrupted by at least one heteroatom.

In a preferred aspect, R¹ is not interrupted by a heteroatom.Alternatively, R¹ is interrupted by at least one ether, thioether,substituted amino, or amido group.

In a preferred aspect, R¹ is C₁-C₂₀ alkyl. In a more preferred aspect,R¹ is C₁-C₁₂ or C₂-C₈ alkyl. In a still more preferred aspect, R¹ is 2,3, or 4.

In another preferred aspect, R¹ is (CH₂)_(r)SO₃H or (CH₂)_(r)SO₃ ⁻; andr is an integer from 1 to 20. In a more preferred aspect, r is 2, 3, or4.

In still another preferred aspect, R¹ is an alkyl group that isadditionally substituted with 1 R¹⁴. In a more preferred aspect, the R¹⁴is carboxy or sulfonato. In a still more preferred aspect, R¹⁴ issulfonato. In a yet still more preferred aspect, R¹⁴ is3-sulfonatopropyl or 4-sulfonatobutyl.

In yet another preferred aspect, R¹ is an unbranched alkyl group that isadditionally substituted with 1 R¹⁴. In a more preferred aspect, R¹ isan unbranched alkyl group that is substituted with R¹⁴ at the end of thealkyl group opposite to its attachment point to the cyanine dyeheterocyclic nitrogen. In a still more preferred aspect, R¹ is2-sulfonatoethyl, 3-sulfonatopropyl, 4-sulfonatobutyl, or5-sulfonatopentyl. In a yet still more preferred aspect, R¹ is3-sulfonatopropyl or 4-sulfonatobutyl; more preferably, R¹ is3-sulfonatopropyl.

Each R^(2a) and R^(2b) is a member independently selected from the groupconsisting of alkyl, alkenyl, hydroxyalkyl, alkoxyalkyl, aminoalkyl,amidoalkyl, alkylthioalkyl, carboxyalkyl, alkoxycarbonylalkyl, orsulfonatoalkyl; wherein a carbon of the member is additionallysubstituted with from 0 to 1 -L-Y—Z; or, alternatively, a R^(2a) andR^(2b) pair, together with the ring carbon to which the R^(2a) andR^(2b) are bonded, join to form a spirocycloalkyl ring, wherein thespirocycloalkyl ring is additionally substituted with from 0 to 6 R¹⁴and from 0 to 1 -L-Y—Z, or an exocyclic alkene, wherein the exocyclicalkene is additionally substituted with from 0 to 2 R¹⁴ and from 0 to 1-L-Y—Z.

In a preferred aspect, all R^(2a) are the same substituent.Alternatively, all R^(2b) are the same substituent. More preferably, allR^(2a) are the same substituent, and all R^(2b) are the samesubstituent.

In another preferred aspect, R^(2a) and R^(2b) are the same. In a morepreferred aspect, R^(2a) and R^(2b) are alkyl, alkenyl, aminoalkyl,carboxyalkyl, or sulfonatoalkyl. In a still more preferred aspect,R^(2a) and R^(2b) are alkyl, carboxyalkyl, or sulfonatoalkyl. In a yetstill more preferred aspect, R^(2a) and R^(2b) are methyl.

In an alternative aspect, R^(2a) and R^(2b) are different. In a morepreferred aspect, R^(2a) is alkyl, and R^(2b) is selected from the groupof alkyl, alkenyl, aminoalkyl, carboxyalkyl, or sulfonatoalkyl. In astill more preferred aspect, R^(2a) is alkyl, and R^(2b) is selectedfrom the group of alkyl, carboxyalkyl, or sulfonatoalkyl. Yet still morepreferably, R^(2a) is methyl.

In another preferred aspect, R^(2a) and R^(2b), together with the ringcarbon to which R^(2a) and R^(2b) are bonded, join to form aspirocycloalkyl ring, wherein the spirocycloalkyl ring is additionallysubstituted with from 0 to 6 R¹⁴. In a more preferred aspect, R^(2a) andR^(2b) form a cyclopentyl or sulfonatoalkyl. In a still more preferredaspect, R¹⁴ is alkyl. In a yet still more preferred aspect, R¹⁴ ismethyl.

In an alternative aspect, R^(2a) and R^(2b), together with the ringcarbon to which R^(2a) and R^(2b) are bonded, join to form an exocyclicalkene, wherein the exocyclic alkene is additionally substituted withfrom 0 to 2 R¹⁴. In a more preferred aspect, the exocyclic alkene issymmetrically substituted (e.g., unsubstituted; dialkyl; dicyano).Alternatively, the exocyclic alkene is substituted with two R¹⁴ groups.Still more preferably, the exocylic alkene's R¹⁴ substituent is cyano.

In an alternative preferred aspect, R^(2a) and R^(2b), together with theatom to which R^(2a) and R^(2b) are bonded, join to fowl aspirocycloalkyl ring. In a more preferred aspect, R^(2a) and R^(2b) forma cyclopentyl or cyclohexyl ring. In an alternative more preferredaspect, R^(2a) and R^(2b) form a cyclopentyl or cyclohexyl ringadditionally substituted with from 0 to 6 R¹⁴. In a still more preferredaspect, R¹⁴ is alkyl.

Alternatively and preferably, the spirocycloalkyl ring has at least onepair of geminal R¹⁴ alkyl substituents. More preferably, these geminalR¹⁴ substituents are methyl (e.g., 3,3- or 4,4-dimethyl substitution).

Each R³, R⁴, R⁵, and R⁶ is a member independently selected from thegroup consisting of hydrogen, alkyl, alkenyl, halo, hydroxyl, alkoxy,amino, cyano, carboxyl, alkoxycarbonyl, amido, sulfonato, alkoxyalkyl,carboxyalkyl, alkoxycarbonylalkyl, and sulfonatoalkyl; wherein a carbonof the member is additionally substituted with from 0 to 1 -L-Y—Z; or,alternatively, a pair of said members that is selected from the groupconsisting of R³ and R⁴, R⁴ and R⁵, and R⁵ and R⁶, together with thepair of atoms to which the pair of said members is bonded, joins to forman aryl ring, wherein the aryl ring is additionally substituted withfrom 0 to 2 R¹⁴ and from 0 to 1 -L-Y—Z.

In a first aspect, each R³, R⁴, R⁵, and R⁶ is a member independentlyselected from the group consisting of hydrogen, alkyl, alkenyl, halo,alkoxy, cyano, carboxyl, alkoxycarbonyl, amido, sulfonato, alkoxyalkyl,carboxyalkyl, alkoxycarbonylalkyl, and sulfonatoalkyl. In a preferredaspect, each R³, R⁴, R⁵, and R⁶ is a member independently selected fromthe group of hydrogen, alkyl, carboxy, carboxyalkyl, sulfanato, andsulfanatoalkyl. In a more preferred embodiment, each R³, R⁴, R⁵, and R⁶is a member independently selected from the group of hydrogen andsulfanato.

In one aspect, at least one pair of R³, R⁴, R⁵, or R⁶ are the same(i.e., the R^(n) substituent is not independently selected, but is thesame as the other R^(n) substituent). This aspect can be combined withother aspects specifying the number or type of dye substituents (e.g.,exactly two members of the groups R³, R⁴, R⁵, and R⁶ are hydrogen, andthe two members are the pair of R⁴s). Alternatively, at least two, atleast three, or all four pairs of R³, R⁴, R⁵, or R⁶ are the same. Morepreferably, the dye is symmetric or pseudo-symmetric (i.e., R¹, R^(2a),and R^(2b) are also not independently selected).

In an alternative aspect, at least one member of the groups R³, R⁴, R⁵,and R⁶ is hydrogen. Alternatively, exactly one member of the groups R³,R⁴, R⁵, and R⁶ is hydrogen. In a preferred aspect, at least one pair ofsubstituents selected from the pairs R³ and R⁴; R³ and R⁵; R³ and R⁶; R⁴and R⁵; R⁴ and R⁶; and R⁵ and R⁶ is hydrogen. Alternatively, exactlytwo, exactly three, exactly four, exactly five, or exactly six membersof the groups R³, R⁴, R⁵, and R⁶ are hydrogen. In another aspect,exactly four members of the groups R³, R⁴, R⁵, and R⁶ are hydrogen.Alternatively, exactly five members of the groups R³, R⁴, R⁵, and R⁶ arehydrogen. In a still more preferred aspect, R³, R⁴, and R⁶ are hydrogen.

In another alternative aspect, at least one member of the groups R³, R⁴,R⁵, and R⁶ is sulfonato or sulfonatoalkyl. Alternatively, exactly onesubstituent selected from the groups R³, R⁴, R⁵, and R⁶ is sulfonato orsulfonatoalkyl. In a preferred aspect, R⁵ is sulfonato. In still anotheraspect, both members of a pair of substituents selected from the pairsR³ and R⁴; R³ and R⁵; R³ and R⁶; R⁴ and R⁵; R⁴ and R⁶; and R⁵ and R⁶ areeach a member independently selected from the group of sulfonato orsulfonatoalkyl. Alternatively, exactly two, exactly three, exactly four,exactly five, or exactly six members of the groups R³, R⁴, R⁵, and R⁶are each a member independently selected from the group of sulfonato orsulfonatoalkyl.

In another alternative aspect, at least one member of the group R³, R⁴,R⁵, and R⁶ is anionic at physiological pH (e.g., sulfonato —SO₃ ⁻,carboxy —CO₂ ⁻). Alternatively, exactly one member of the group R³, R⁴,R⁵, and R⁶ is anionic at physiological pH. In a preferred aspect, R⁵ isanionic at physiological pH. In still another aspect, each member of apair of substituents selected from the pairs R³ and R⁴; R³ and R⁵; R³and R⁶; R⁴ and R⁵; R⁴ and R⁶; and R⁵ and R⁶ is anionic at physiologicalpH. Alternatively, exactly two, exactly three, exactly four, exactlyfive, or exactly six members of the groups R³, R⁴, R⁵, and R⁶ areanionic at physiological pH. Alternatively, exactly two, exactly three,or exactly four members of the groups R³, R⁴, R⁵, and R⁶ are anionic atphysiological pH.

In another alternative aspect, at least one member of the groups R³, R⁴,R⁵, and R⁶ is halo. Alternatively, exactly one substituent selected fromthe groups R³, R⁴, R⁵, and R⁶ is halo. In still another aspect, bothmembers of a pair of substituents selected from the pairs R³ and R⁴; R³and R⁵; R³ and R⁶; R⁴ and R⁵; R⁴ and R⁶; and R⁵ and R⁶ are each anindependently selected halo. Alternatively, exactly two, exactly three,exactly four, exactly five, or exactly six members of the groups R³, R⁴,R⁵, and R⁶ are each an independently selected halo.

In a second aspect, a pair of members that is selected from the groupsof R³ and R⁴; R³ and R⁵; R³ and R⁶; R⁴ and R⁵; R⁴ and R⁶; and R⁵ and R⁶,together with the pair of atoms to which the pair of members is bonded,joins to form an aryl ring (i.e., the aryl ring formed from R^(n) andR^(n+1)), wherein the aryl ring is additionally substituted with from 0to 2 R¹⁴. In a preferred aspect, the pair of members R⁵ and R⁶, togetherwith the pair of atoms to which the pair of members is bonded, joins toform a phenyl ring that is additionally substituted with from 0 to 2R¹⁴. In a more preferred aspect, the phenyl ring is additionallysubstituted with from 1 to 2 R¹⁴. In a still more preferred aspect, thephenyl ring is additionally substituted with 2 R¹⁴.

In a preferred aspect, the R¹⁴ substituents of the aryl ring formed fromW and R^(n+1) (e.g., the aryl ring formed from R⁵ and R⁶) are carboxy,carboxyalkyl, sulfonato, or sulfonatoalkyl. In a still more preferredaspect, the R¹⁴ substituents are sulfonato or sulfonatoalkyl. In a yetstill more preferred aspect, the benzindolinium R¹⁴ substituents aresulfonato. In an alternative preferred aspect, the benzindolinium R¹⁴substituents are cyano.

Alternatively, in a preferred aspect, the compound has Formula Ia:

In a more preferred aspect, the aryl ring formed from R^(n) and R^(n−1)is additionally substituted with from 1 to 2 R¹⁴, and a R¹⁴ substituentof the aryl ring is attached to a carbon adjacent to the ring junctionwith the indolinium ring. Alternatively, the aryl ring is additionallysubstituted with from 1 to 2 R¹⁴, and a R¹⁴ substituent of the aryl ringis attached to a carbon non-adjacent to the ring junction with theindolinium ring. Alternatively, the aryl ring is additionallysubstituted with one adjacent substituent and one non-adjacentsubstituent (e.g., the compound of Formula Ib).

Alternatively, in a preferred aspect, the compound has Formula Ib:

In a still more preferred aspect, the benzindolinium R¹⁴ substituents ofFormula Ia or Ib are carboxy, carboxyalkyl, sulfonato, orsulfonatoalkyl. In a still more preferred aspect, the benzindolinium R¹⁴substituents are sulfonato or sulfonatoalkyl. In a yet still morepreferred aspect, the benzindolinium R¹⁴ substituents are sulfonato.

Each R⁷ is a member selected from the group of hydrogen and alkyl; or,alternatively, both R⁷, together with the intervening segment of thepolyene to which both R⁷ are bonded, join to form a ring, wherein thering is selected from the group of a cycloalkyl and a heterocyclyl ring,and wherein the ring is additionally substituted with from 0 to 3 R¹⁴and from 0 to 1 -L-Y—Z.

In one aspect, both R⁷, together with the intervening segment of thepolyene to which both R⁷ are bonded, join to form a ring selected fromthe group of a five-membered ring and a six-membered ring, wherein thering is additionally substituted with from 0 to 3 R¹⁴. In a morepreferred aspect, the ring is a six-membered ring (e.g., both R⁷ combineto form a propylidene linking group). In a still more preferred aspect,the ring is cyclohexyl (i.e., both R⁷ combine to form a —(CH₂)₃— linkinggroup). In an alternative more preferred aspect, the ring is afive-membered ring. In another still more preferred aspect, the ring iscyclopentyl (i.e., both R⁷ combine to form a —(CH₂)₂— linking group).

R⁸, R⁹, R¹⁰, R¹¹ and R¹² are each a member independently selected fromthe group consisting of hydrogen, alkyl, alkenyl, halo, alkoxy,sulfonato, hydroxyl, amino, carboxyl, alkoxycarbonyl, cyano, amido,thioacetyl, and -L-Y—Z; wherein at least one member selected from thegroup consisting of R⁸, R⁹, and R¹⁰ is -halo. More preferably, at leastone member selected from the group consisting of R⁸, R⁹, and R¹⁰ isfluoro or chloro.

In one aspect, R⁸ is halo; more preferably, fluoro or chloro.Preferably, R⁹, R¹⁰, R¹¹, and R¹² are each a member independentlyselected from the group consisting of hydrogen, alkyl, alkoxy, halo,sulfonato, and -L-Y—Z.

In a second aspect, R⁸ is hydrogen, alkyl, alkoxy, or halo. In anothermore preferred aspect, R⁸ is fluoro; alternatively, R⁸ is chloro.

In an alternative preferred aspect, R⁸ is hydrogen.

Alternatively, R⁸ is a carboxyalkyl. Preferably, R⁸ is a lower alkylgroup with a carboxy-substituent. More preferably, R⁸ is5-carboxypentyl, 4-carboxybutyl, 3-carboxypropyl, 2-carboxyethyl, orcarboxymethyl. Still more preferably, R⁸ is 5-carboxypentyl or2-carboxyethyl.

Alternatively, R⁸ is carboxyl, alkoxycarbonyl, or amido; morepreferably, R⁸ is carboxyl.

In one aspect, R¹⁰ is halo; more preferably, fluoro or chloro.Preferably, R⁸, R⁹, R¹¹, and R¹² are each a member independentlyselected from the group consisting of hydrogen, alkyl, alkoxy, halo,sulfonato, and -L-Y—Z.

In a second aspect, R¹⁰ is hydrogen, alkyl, alkoxy, or halo. In anothermore preferred aspect, R¹⁰ is fluoro; alternatively, R¹⁰ is chloro.

In an alternative preferred aspect, R¹⁰ is hydrogen.

Alternatively, R¹⁰ is a carboxyalkyl. Preferably, R¹⁰ is a lower alkylgroup with a carboxy-substituent. More preferably, R¹⁰ is5-carboxypentyl, 4-carboxybutyl, 3-carboxypropyl, 2-carboxyethyl, orcarboxymethyl. Still more preferably, R¹⁰ is 5-carboxypentyl or2-carboxyethyl.

Alternatively, R¹⁰ is carboxyl, alkoxycarbonyl, or amido; morepreferably, R¹⁰ is carboxyl.

In one aspect, R⁹ is -L-Y—Z. Preferably, R⁸, R¹⁰, R¹⁴, and R¹² are eacha member independently selected from the group consisting of hydrogen,alkyl, alkoxy, halo, sulfonato, and -L-Y—Z.

In a second aspect, R⁹ is hydrogen, alkyl, alkoxy, or halo. In anothermore preferred aspect, R⁹ is fluoro; alternatively, R⁹ is chloro.

In an alternative preferred aspect, R⁹ is hydrogen.

Alternatively, R⁹ is a carboxyalkyl. Preferably, R⁹ is a lower alkylgroup with a carboxy-substituent. More preferably, R⁹ is5-carboxypentyl, 4-carboxybutyl, 3-carboxypropyl, 2-carboxyethyl, orcarboxymethyl. Still more preferably, R¹⁰ is 5-carboxypentyl or2-carboxyethyl.

Alternatively, R⁹ is carboxyl, alkoxycarbonyl, or amido; morepreferably, R⁹ is carboxyl.

In one aspect, R¹¹ and R¹² are each a member independently selected fromthe group of hydrogen, alkyl, alkenyl, halo, alkoxy, sulfonato,hydroxyl, amino, carboxyl, alkoxycarbonyl, cyano, amido, thioacetyl, and-L-Y—Z. Preferably, R¹¹ and R¹² are each a member independently selectedfrom the group of hydrogen, alkyl, halo, and sulfonato. More preferably,R¹¹ and R¹² are each a member independently selected from the group ofhydrogen, halo, and sulfonato.

In a second aspect, R¹¹ is hydrogen, alkyl, alkoxy, or halo. In a morepreferred aspect, R¹¹ is halo; preferably, R¹¹ is fluoro or chloro. Inan alternative aspect, R¹¹ is hydrogen. Alternatively, R¹⁰ and R¹¹ arehydrogen.

In a third aspect, R¹² is hydrogen, alkyl, alkoxy, or halo. In a morepreferred aspect, R¹² is halo; preferably, R¹² is fluoro or chloro. In astill more preferred aspect, R¹⁰ and R¹² are halogen, preferably fluoroor chloro. Alternatively, R¹¹ and R¹² are halogen, preferably fluoro orchloro. In a yet still more preferred aspect, R¹⁰, R¹¹, and R¹² arehalogen, preferably fluoro or chloro.

In a fourth aspect, the phenyl ring substituted with R⁸, R⁹, R¹⁰, R¹¹,and R¹² is 1,2,3-substituted with independently selected substituentsother than hydrogen, wherein the 1-substituent is the polymethine bridge(e.g., R⁸ and R⁹ are the same halo group; R⁸ and R⁹ are different halogroups; R⁸ is halo- and R⁹ is -L-Y—Z). Alternatively, the ring is1,2,4-substituted. Alternatively, the ring is 1,2,5-substituted.Alternatively, the ring is 1,2,6-substituted. Alternatively, the ring is1,3,4-substituted. Alternatively, the ring is 1,3,5-substituted.Alternatively, the ring is 1,3,6-substituted. Preferably, at least twoof the substituents are halo; more preferably. fluoro or chloro.

In a fifth aspect, the phenyl ring substituted with R⁸, R⁹, R¹⁰, R¹¹,and R¹² is 1,2,3,4-substituted with independently selected substituentsother than hydrogen, wherein the 1-substituent is the polymethinebridge. Alternatively, the ring is 1,2,3,5-substituted. Alternatively,the ring is 1,2,3,6-substituted. Alternatively, the ring is1,2,4,5-substituted. Alternatively, the ring is 1,2,4,6-substituted.Alternatively, the ring is 1,2,5,6-substituted. Alternatively, the ringis 1,3,4,5-substituted. Alternatively, the ring is 1,3,4,6-substituted.Alternatively, the ring is 1,3,5,6-substituted. Preferably, at least twoof the substituents are halo; more preferably, fluoro or chloro.Alternatively and preferably, at least three of the substituents arehalo; more preferably, fluoro or chloro.

In a sixth aspect, the phenyl ring substituted with R⁸, R⁹, R¹⁰, R¹¹,and R¹² is 1,2,3,4,5-substituted with independently selectedsubstituents other than hydrogen, wherein the 1-substituent is thepolymethine bridge. Alternatively, the ring is 1,3,4,5,6-substituted.Alternatively, the ring is 1,2,4,5,6-substituted. Alternatively, thering is 1,2,3,5,6-substituted. Alternatively, the ring is1,2,3,4,6-substituted. Alternatively, the ring is independentlysubstituted at each ring position.

Preferably, at least two of the substituents are halo; more preferably.fluoro or chloro. Alternatively, at least three of the substituents arehalo; more preferably. fluoro or chloro (e.g., R⁸, R¹⁰, and R¹² arehalo). Alternatively, at least four of the substituents are halo; morepreferably. fluoro or chloro (e.g., R⁸, R⁹, R¹¹, and R¹² are halo).

In a seventh aspect, R⁸, R⁹, R¹⁰, R¹¹, and R¹² are each a memberindependently selected from the group of hydrogen, alkyl, alkoxy, halo,sulfonato, and sulfonatoalkyl.

Fluoro substitution has been shown to increase quantum yield andphotostability in fluorescein dyes as well as lowering dye pK_(a). SeeSun, W.-C. et al. J. Org. Chem. 1997, 62, 6469-6475. In an eighthaspect, at least one member of the group R⁸, R⁹, R¹⁰, R¹¹, and R¹² is afluoro substituent. Alternatively, at least one member of the groups R³,R³, R⁴, R⁵, R⁶, and R¹⁴ is a fluoro substituent. In yet another aspect,at least two members of the groups R³, R⁴, R, R⁶, R⁸, R⁹, R¹⁰, R¹², andR¹⁴ are fluoro substituents. In yet another aspect, at least threemembers of R³, R⁴, R, R⁶, R⁸, R⁹, R¹⁰, R¹¹, R¹², and R¹⁴ are fluorosubstituents. In yet another aspect, at least four or at least fivemembers of the groups R³, R⁴, R, R⁶, R⁸, R⁹, R¹⁰, R¹¹, R¹², and R¹⁴ arefluoro substituents.

As demonstrated in the examples, chloro substitution also has afavorable effect on the dye properties, much as the fluoro group does.In a ninth aspect, at least one member of the group R⁸, R⁹, R¹⁰, R¹¹,and R¹² is a chloro substituent. Alternatively, at least one member ofthe groups R³, R⁴, R⁵, R⁶, R⁸, R⁹, R¹⁰, R¹¹, R¹², and R¹⁴ is a chlorosubstituent. In yet another aspect, at least two members of the groupsR³, R⁴, R⁵, R⁶, R⁸, R⁹, R¹⁰, R¹¹, R¹², and R¹⁴ are chloro substituents.In yet another aspect, at least three members of the groups R³, R⁴, R,R⁶, R⁸, R⁹, R¹⁰, R¹¹, R¹², and R¹⁴ are chloro substituents. In yetanother aspect, at least four or at least five members of the groups R³,R⁴, R, R⁶, R⁸, R⁹, R¹⁰, R¹¹, R¹², and R¹⁴ are chloro substituents.

Each R¹³ is a member independently selected from the group of hydroxyl,amino, carboxyl, and alkoxycarbonyl. In a preferred embodiment, R¹³ iscarboxyl or alkoxycarbonyl. In a more preferred embodiment, R¹³ iscarboxyl. Alternatively, R¹³ is cyano.

Each R¹⁴ is a member independently selected from the group consisting ofalkyl, alkenyl, halo, hydroxyl, alkoxy, amino, amido, amidoalkyl, cyano,cyanoalkyl, carboxyl, alkoxycarbonyl, amido, sulfonato, sulfonatoalkyl,thioacetyl, thioacetylalkyl, alkoxycarbonylalkyl, and alkoxyalkyl;wherein the alkyl or alkenyl is additionally substituted with from 0 to1 R¹³ and from 0 to 1 -L-Y—Z. In a preferred aspect, R¹⁴ is alkyl,alkenyl, carboxyl, alkoxycarbonyl, amido, alkoxycarbonylalkyl, halo,sulfonato, or sulfonatoalkyl. Alternatively, R¹⁴ is carboxyalkyl,hydroxyalkyl, halo, sulfonato, or sulfonatoalkyl. In an alternativeaspect, R¹⁴ is alkyl or alkyl substituted with 1 R¹³. Alternatively, R¹⁴is halo or sulfonato. Alternatively, R¹⁴ is sulfonato.

Each L is an optional member independently selected from the groupconsisting of a bond, a C₁-C₂₀ alkylene, and a C₁-C₂₀ alkenylene;wherein the alkylene or alkenylene is optionally interrupted by at leastone heteroatom. In a preferred aspect, L is a bond, with the provisothat L is not a bond when that would produce a highly unstable structure(e.g., N-L-R¹³, if R¹³ is —CO₂H). Alternatively, L is a C₁-C₁₄ alkylene;more preferably, L is a C₁-C₁₀ alkylene or a C₁-C₆ alkylene.Alternatively, L is a C₁-C₁₂ alkylene interrupted by ether linkages(e.g., a polyethylene glycol oligomer).

In a preferred aspect, the alkylene or alkenylene is not interrupted bya heteroatom. Alternatively, L is interrupted by at least one ether,thioether, substituted amino, or amido group.

Each Y is an optional member independently selected from the groupconsisting of a bond, —O—, —S—, —NH—, —NHC(O)—, —C(O)NH—, —NR¹⁵—,—NR¹⁵C(O)—, —C(O)NR¹⁵—, —NZ—, —NZC(O)—, and —C(O)NZ—. In a preferredaspect, Y is a bond. Alternatively, Y is —O—. Alternatively, Y is anamido group optionally substituted with R¹⁵ at the amido nitrogen.

Each Z is independently selected from the group consisting of R¹³ andR¹⁶. In a more preferred aspect, Z is C₁-C₆ alkyl. Alternatively, Z isinterrupted by ether linkages (e.g., a polyethylene glycol oligomer). Ina still more preferred aspect, Z is carboxyalkyl or alkyl with anactivated acyl substituent. In a yet still more preferred aspect, Z is5-carboxypentyl or 4-carboxybutyl.

In an alternative preferred aspect, Z is a carboxyalkyl. Preferably, Zis a lower alkyl group with a carboxy-substituent. More preferably, Z is5-carboxypentyl, 4-carboxybutyl, 3-carboxypropyl, 2-carboxyethyl, orcarboxymethyl. Still more preferably, Z is 5-carboxypentyl or2-carboxyethyl.

In another alternative preferred aspect, -L-Y— is (CH₂)_(t); Z iscarboxyl or activated acyl; and t is an integer from 0 to 10.

In still another alternative preferred aspect, the Z group's L group isa bond, and R¹³ or R¹⁶ is connected directly to -L-Y— or directly bondedto the phenyl ring itself if L and Y are also bonds.

In yet still another alternative preferred aspect, -L-Y—Z has at leastthreecarbons. Alternatively, Z has at least three carbons.

In yet still another alternative preferred aspect, -L-Y—Z has at leastfour carbons. Alternatively, Z has at least four carbons.

In an alternative embodiment, —Y—Z is a member selected from the groupconsisting of —N(Z)₂, —N(Z)C(O)Z, and —C(O)N(Z)₂, and the two Z groupsmay optionally be linked to form a cycloalkynyl group. Examples of—N(Z)₂ cycloalkynyl groups are DBCO or DBCO-1, which are shown below:

Each R¹⁴ is a member independently selected from the group of alkyl,alkenyl, halo, hydroxyl, alkoxy, amino, amido, amidoalkyl, cyano,cyanoalkyl, carboxyl, alkoxycarbonyl, amido, sulfonato, sulfonatoalkyl,thioacetyl, thioacetylalkyl, alkoxycarbonylalkyl, and alkoxyalkyl;wherein the R¹⁴ alkyl or alkenyl is additionally substituted with from 0to 1 R¹³. In a preferred aspect, R¹⁴ is alkyl, alkenyl, carboxyl,alkoxycarbonyl, amido, or alkoxycarbonylalkyl. Alternatively, R¹⁴ issulfonato. In a more preferred aspect, R¹⁴ is alkyl or alkyl substitutedwith 1 R¹³. Alternatively, R¹⁴ is carboxyalkyl, hydroxyalkyl, orsulfonatoalkyl.

Each R¹⁵ is a member independently selected from the group consisting ofalkyl and alkoxycarbonylalkyl; wherein the alkyl is optionallyinterrupted by at least one heteroatom.

In a preferred aspect, the alkyl is not interrupted by a heteroatom. Ina preferred aspect, R¹⁵ is alkyl. In a more preferred aspect, R¹⁵ islower alkyl.

Alternatively, L is interrupted by at least one ether, thioether,substituted amino, or amido group. Preferably, R¹⁵ is interrupted by atleast one ether group (e.g., a polyethylene glycol oligomer).

Each R¹⁶ is independently a member selected from the group consisting ofactivated acyl, acrylamido, optionally substituted alkylsulfonate ester,azido, optionally substituted arylsulfonate ester, optionallysubstituted amino, aziridino, boronato, cycloalkynyl,cycloalkynylcarbonyl, diazo, formyl, glycidyl, halo, haloacetamidyl,haloalkyl, haloplatinato, halotriazino, hydrazinyl, imido ester,isocyanato, isothiocyanato, maleimidyl, mercapto, phosphoramidityl, aphotoactivatable moiety, vinyl sulfonyl, alkynyl, a pegylated azido, apegylated alkynyl, a pegylated cycloalkynyl, an ortho substitutedphosphinyl aryl ester (e.g., TPPME), a spirocycloalkynyl, and an orthosubstituted phosphine oxide aryl ester.

In one aspect, each R¹⁶ is independently a member selected from thegroup consisting of activated acyl, acrylamido, optionally substitutedalkylsulfonate ester, alkynyl, optionally substituted arylsulfonateester, amino, azido, aziridino, boronato, diazo, formyl, glycidyl, halo,haloacetamidyl, haloalkyl, haloplatinato, halotriazino, hydrazinyl,imido ester, isocyanato, isothiocyanato, maleimidyl, mercapto,phosphoramidityl, a photoactivatable moiety, and vinyl sulfonyl. In apreferred aspect, R¹⁶ is activated acyl, maleimidyl, phosphoramidityl,or glycidyl. In a more preferred embodiment, R¹⁶ is activated acyl.Alternatively, R¹⁶ is activated ester. In a still more preferredembodiment, R¹⁶ is succinimidyloxy-ester or sulfosuccinimidyloxy-ester.

In certain aspects, R¹⁶ has the following structures:

In a preferred aspect, R¹⁶ is activated acyl, maleimidyl,phosphoramidityl, or glycidyl. In a more preferred embodiment, R¹⁶ isactivated acyl. Alternatively, R¹⁶ is activated ester. In a still morepreferred embodiment, R¹⁶ is succinimidyloxy-ester orsulfosuccinimidyloxy-ester.

The compound has a balanced charge. In a preferred aspect, thecompound's net anionic charge is balanced by alkali metal counterions(e.g., sodium or potassium). In a more preferred aspect, at least one ofthe counterions is sodium. Alternatively, all of the counterions aresodium.

In an alternative embodiment, the compound has a balanced charge inwhich positively and negatively charged substituents are balanced sothat the dye molecule has a net charge of −1, 0, or +1 (preferably, 0),even without its counterions (i.e., the dye counterion has a net chargeof −1, 0, or +1). In some aspects, this net charge is produced byincluding numbers of positively and negatively charged substituentgroups that produce a dye net charge of −1, 0, or +1. This type ofcharge balancing is discussed in U.S. Provisional Application 61/150,522(filed Feb. 9, 2009) and WO 2010/091243 (filed Feb. 5, 2010), which areincorporated by reference.

In a preferred aspect, -L-Y—Z is —(CH₂)₄CO₂H or an ester derivativethereof. Alternatively, -L-Y—Z is —(CH₂)₂CO₂H or an ester derivativethereof. Preferably, each R³, R⁴, R⁵, and R⁶ are a member independentlyselected from the group of hydrogen, alkyl, halo, and sulfonato.

In certain aspects, an activated acyl group is present in place of thecarboxy group. In a still more preferred aspect, the activated acylgroup is an activated ester. In a still yet more preferred aspect, theactivated ester is a succinimidyloxy-ester.

In a first aspect, the compound of Formula I, Ia, Ib, II, IIa, or IIbfluoresces at a wavelength within the range of about 550 nm to about1000 nm. Preferably, the compound fluoresces at a wavelength within therange of about 600 nm to about 850 nm. More preferably, the compoundfluoresces at a wavelength within the range of about 600 nm to about 725nm. Alternatively, the compound fluoresces at a wavelength within therange of about 725 nm to about 850 nm.

Alternatively, the compound of Formula I, Ia, Ib, II, IIa, or IIbfluoresces at a wavelength within the range of about 600 nm to about1000 nm. Preferably, the compound fluoresces at a wavelength within therange of about 600 nm to about 725 nm. Alternatively, the compoundfluoresces at a wavelength within the range of about 650 nm to about 850nm. Alternatively, the compound fluoresces at a wavelength within therange of about 725 nm to about 850 nm.

One preferred aspect of the instant invention is compounds with the samesubstituents on both heterocyclic rings (e.g., both R¹ are the samesulfonatoalkyl substituent, optionally with different counterions tobalance charge). This provides advantages during the synthesis andpurification of the compound.

The present application broadly encompasses all possible stereoisomersof the compounds as described herein, including the variousdiasteromers, enantiomers, and olefin stereoisomers apparent to one ofskill in the art. This application is further directed to all methods ofpurifying cyanine dye compound stereoisomers that are well-known in theart as well as the purified compounds available by these methods.

Preparation of Compounds of Formula I

In one aspect, the preferred cyanine compounds set forth in pending U.S.patent application Ser. No. 12/065,391 (US 2008/0267883 A1). Arepresentative procedure for a Schiff base is included in U.S. Pat. No.6,747,159 (Ar=Ph; pyridine/Ac₂O, Δ). The substituent can optionally bemodified after the synthesis of the polymethine bridge (e.g.,deprotected, activated for reaction with a biomolecule, or reacted toform a linking group).

In another aspect, the preferred cyanine compounds of Formula I, Ia, orIb are prepared by means of an organometallic coupling to incorporate asubstituent to the polymethine bridge. More preferably, the substituentis installed by means of a palladium coupling. The substituent canoptionally be modified after its inclusion (e.g., deprotected, activatedfor reaction with a biomolecule, or reacted to form a linking group).

The Miyaura-Suzuki reaction, also known as the Suzuki coupling, has beenextensively used in organic synthesis since its discovery: Miyaura, N.;Yamada, K.; Suzuki, A. Tetrahedron Lett. 1979, 36, 3437-3440. Recently aSuzuki coupling was used to install a substituted aryl substituent atthe central position of a heptamethine bridge in a water-soluble cyaninedye: Lee, H.; Mason, J. C.; Achilefu, S. J. Org. Chem. 2006, 71,7862-7865.

However, because many cyanine dyes decompose under standard Suzukicoupling conditions of heating with a base, few examples of its use forthe synthesis of cyanine dyes are known.

In a particularly preferred aspect of the instant invention, thesubstituent of a compound of Formula I is incorporated by means of aSuzuki coupling reaction, some of which are detailed in the examples ofthis specification. In one embodiment, the polymethine substrate for theSuzuki coupling is a 3-halopentamethine or a 4-haloheptamethine. In apreferred embodiment, the halo-substituent is a chloride or a bromide.In a more preferred embodiment, the halo-substituent is a bromide.

Other means of preparing cyanine dyes and their synthetic precursors areincluded in Hamer, F. M., Cyanine Dyes and Related Compounds,Weissberger, Mass., ed. Wiley Interscience, N.Y. 1964; and Mojzych, M.,Henary, M. “Synthesis of Cyanine Dyes,” Top. Heterocycl. Chem., vol. 14,Springer Berlin, Heildelberg, 2008, pp. 1-9. Further, U.S. Pat. Nos.4,337,063; 4,404,289; and 4,405,711 describe a synthesis for a varietyof cyanine dyes having N-hydroxysuccinimide active ester groups. U.S.Pat. No. 4,981,977 describes a synthesis for cyanine dyes havingcarboxylic acid groups. U.S. Pat. No. 5,268,486 discloses a method formaking arylsulfonate cyanine dyes. U.S. Pat. No. 6,027,709 disclosesmethods for making cyanine dyes having phosphoramidite groups. U.S. Pat.No. 6,048,982 discloses methods for making cyanine dyes having areactive group selected from the group of isothiocyanate, isocyanate,phosphoramidite, monochlorotriazine, dichlorotriazine, mono- ordi-halogen substituted pyridine, mono- or di-halogen substituteddiazine, aziridine, sulfonyl halide, acid halide, hydroxysuccinimideester, hydroxy sulfosuccinimide ester, imido ester, glyoxal andaldehyde.

One common synthetic route involves preparing substituted orunsubstituted indolesulfonate quaternary salts according to proceduresthat are well-known in the art, some of which are detailed in theexamples of this specification. Particularly preferred indole quaternarysalts include, among others, indolesulfonate and benzindolesulfonatequaternary salts, which are exemplified in this specification.

The pair of synthesized salts are then reacted with a dialdehyde or adialdehyde equivalent (e.g., a Schiff base) to form the polymethinebridge by means of techniques and reaction conditions that arewell-known in the art, some of which are detailed in the examples ofthis specification. Preferably, one of the dialdehydes is protected ormasked to allow incorporation of one polycyclic side of the bridge(e.g., the indoline ring), followed by deprotection or unmasking of thealdehyde and by incorporation or construction of the other polycyclicgroup (e.g., the pyrrolopyridine). Schiff bases can be purchased fromcommercial suppliers (e.g., Sigma-Aldrich) or prepared according toprocedures that are well-known in the art (e.g., the method of Example5).

Methods of Labeling Biomolecules

The cyanine compounds of Formula I can be attached to biomolecules,which are defined above. Methods of linking dyes to various types ofbiomolecules are well-known in the art. For a through review of, e.g.,oligonucleotide labeling procedures, see R. Haugland in Excited Statesof Biopolymers, Steiner ed., Plenum Press (1983), Fluorogenic ProbeDesign and Synthesis: A Technical Guide, PE Applied Biosystems (1996),and G. T. Herman, Bioconjugate Techniques, Academic Press (1996).

“Click” chemistry provides one possible way for linking the inventivedyes to biomolecules. Click chemistry uses simple, robust reactions,such as the copper-catalyzed cycloaddition of azides and alkynes, tocreate intermolecular linkages. For a review of click chemistry, seeKolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem. 2001, 40, 2004.

Connection (or ligation) of two fragments to make a larger molecule orstructure is often achieved with the help of so-called “click chemistry”described by Sharpless et al. Angew. Chem., Int. Ed. 40: 2004 (2001).This term is used to describe a set of bimolecular reactions between twodifferent reactants such as azides and acetylenes. The formation of1,2,3-triazoles in 1,3-dipolar cycloaddition of azides to a triple bondis known, but because the activation energy of acetylene-azidecycloaddition is relatively high, the reaction is slow under ambientconditions.

The utility of the reaction of azides with alkynes was expanded by thediscovery of Cu (I) catalysis. 1,3-cycloaddition of azides to terminalacetylenes in the presence of catalytic amounts of cuprous salts isfacile at room temperature in organic or aqueous solutions.

U.S. Pat. No. 7,807,619 to Bertozzi et al. teaches modified cycloalkynecompounds and method of use of such compounds in modifying biomolecules.Bertozzi et al. teach a cycloaddition reaction that can be carried outunder physiological conditions. As disclosed therein, a modifiedcycloalkyne is reacted with an azide moiety on a target biomolecule,generating a covalently modified biomolecule.

The present invention provides cyanine dyes with click chemistryfunctionalities useful for labeling biomolecules. As such, in oneaspect, the present invention provides compounds of Formula I or II, inwhich I one embodiment, each R¹⁶ is independently a member selected fromthe group consisting of activated acyl, acrylamido, optionallysubstituted alkylsulfonate ester, azido, optionally substitutedarylsulfonate ester, amino, azido, aziridino, boronato, diazo, formyl,glycidyl, halo, haloacetamidyl, haloalkyl, haloplatinato, halotriazino,hydrazinyl, imido ester, isocyanato, isothiocyanato, maleimidyl,mercapto, phosphoramidityl, a photoactivatable moiety, vinyl sulfonyl,alkynyl, a pegylated azido group, and a pegylated alkynyl group; and inwhich at least one R¹⁶ is independently a member selected from the groupazido, alkynyl, a pegylated azido and a pegylated alkynyl.

In yet other aspects, the present invention relates to two componentsthat interact with each other to form a stable covalent bio-orthogonalbond. Bio-orthogonal reactions are reactions of materials with eachother, wherein each material has limited or essentially no reactivitywith functional groups found in vivo. These components are of use inchemical and biological assays, as chemical reagents, medical imagingand therapy, and more particularly, in nucleic acid modificationtechniques. According to a particular embodiment of the invention, thecovalent bio-orthogonal bond is obtained by the [3+2]cycloaddition ofazides and alkynes.

In still other aspects, one of the two components that interact witheach other to form a stable covalent bio-orthogonal bond is a nearinfrared dye, such as a cyanine dye. In a preferred aspect, the cyaninedyes of the present invention comprise either an azide or an alkynegroup for use as a reactant in a click chemistry reaction and the otherreactant is a biomolecule such as a nucleotide comprising either analkyne or azide group.

Azide reactive groups such as an alkyne compounds can react with atleast one 1,3-dipole-functional compound such as an alkyne reactivegroup (e.g., a azido group) in a cyclization reaction to form aheterocyclic compound. In certain embodiments, the reaction can becarried out in the presence of an added catalyst (e.g., Cu(I)). In otherembodiments, the reaction is carried out in the absence of suchcatalysts. Exemplary 1,3-dipole-functional compounds include, but arenot limited to, azide-functional compounds, nitrile oxide-functionalcompounds, nitrone-functional compounds, azoxy-functional compounds,and/or acyl diazo-functional compounds. Preferably, azide-functionalcompounds are used.

Suitable biomolecule moieties for click reaction include, for example,monomeric and polymeric derivatives of nucleotides, carbohydrates, aminoacids, lipids, glycols, alkanes, alkenes, arene, silicates, as well asbiologically active and inactive compounds obtained from nature or fromartificial synthesis.

Other suitable biological molecules include those having a azido oralkynyl functionality, which include, but are not limited to, anantibody, an antigen, an avidin, a carbohydrate, a deoxy nucleic acid, adideoxy nucleotide triphosphate, an enzyme cofactor, an enzymesubstrate, a fragment of DNA, a fragment of RNA, a hapten, a hormone, anucleic acid, a nucleotide, a nucleotide triphosphate, a nucleotidephosphate, a nucleotide polyphosphate, an oligosaccharide, a peptide,PNA, a polysaccharide, a protein, a streptavidin, and the like. Thesebiological molecules will in turn be reacted with the dye compounds ofthe present invention comprising either an azide or an alkyne group foruse in click chemistry reactions.

In one aspect, the cyanine compounds of Formula I have sufficientsolubility in aqueous solutions that once they are conjugated to asoluble ligand or biomolecule, the ligand or biomolecule retains itssolubility. In certain instances, the bioconjugates also have goodsolubility in organic media (e.g., DMSO or DMF), which providesconsiderable versatility in synthetic approaches to the labeling ofdesired materials.

In another aspect, the present invention provides a method or processfor labeling a ligand or biomolecule with a compound of Formula I, themethod comprising: contacting a ligand or biomolecule with a compoundhaving Formula I, Ia, or Ib to generate the corresponding bioconjugatecompound of Formula II, IIa, or IIb.

In one preferred embodiment, the R¹⁶ group or the R¹³ group reacts witha thiol, a hydroxyl, a carboxyl, or an amino group on a biomolecule,forming a linking group between the dye and the biomolecule. In a morepreferred embodiment, this reaction is carried out in mixtures ofaqueous buffer and an organic solvent such as DMF at pH 8 to 9.Alternatively, this reaction is carried out in distilled water or in anaqueous buffer solution. For thiols or for acidic groups, a pH of 7 orlower is preferred for the reaction solvent, especially if a substratealso contains a reactive amino group.

Selected examples of reactive functionalities useful for attaching acompound of Formula I to a ligand or biomolecule are shown in Table 1,wherein the bond results from the reaction of a dye with a ligand orbiomolecule. Column A of Table 1 is a list of the reactivefunctionalities, which can be on the compound of Formula I or thebiomolecule. Column B is a list of the complementary reactive groups(preferably, a carboxyl, hydroxyl, thiol, or amino functionality), whichcan be on the biomolecule or the compound of Formula I, and which reactwith the indicated functionality of Column A to form the bond of ColumnC. Those of skill in the art will know of other bonds suitable for usein the present invention.

TABLE 1 Exemplary Bonds for Linking Groups B A Complementary C ReactiveFunctionality Group (Biomolecule Resulting (Compound of Formula I orCompound Linking or Biomolecule) of Formula I) Group activated esters*amines/anilines amides acrylamides thiols thioethers acyl azides**amines/anilines amides acyl halides amines/anilines amides acyl halidesalcohols/phenols esters acyl nitriles alcohols/phenols esters acylnitriles amines/anilines amides aldehydes amines/anilines iminesaldehydes or ketones hydrazines hydrazones aldehydes or ketoneshydroxylamines oximes alkyl halides amines/anilines alkyl amines alkylhalides carboxylic acids esters alkyl halides thiols thioethers alkylhalides alcohols/phenols ethers anhydrides alcohols/phenols estersanhydrides amines/anilines amides/imides aryl halides thiols thiophenolsaryl halides amines aryl amines azides alkynes 1,2,3-triazoles azidesester with phosphine amide (and phosphine reagent (e.g., o- oxide)diphenylphosphino group) aziridines thiols thioethers boronates glycolsboronate esters boronates/boronic acids aryl halides C-C bond to arylring boronates/boronic acids alkenyl halides C-C bond to alkenyl groupactivated carboxylic acids amines/anilines amides activated carboxylicacids alcohols esters activated carboxylic acids hydrazines hydrazidescarbodiimides carboxylic acids N-acylureas or anhydrides diazoalkanescarboxylic acids esters electron-rich diene dienophile (e.g.,cyclohexene (Diels- electron-poor Alder cycloaddition) alkene) epoxidesthiols thioethers epoxides amines alkyl amines epoxides carboxylic acidsesters haloacetamides thiols thioethers haloplatinate amino platinumcomplex haloplatinate heterocycle platinum complex halotriazinesamines/anilines aminotriazines halotriazines alcohols/phenols triazinylethers imido esters amines/anilines amidines isocyanates amines/anilinesureas isocyanates alcohols/phenols urethanes isothiocyanatesamines/anilines thioureas maleimides thiols thioethers phosphoramiditesalcohols phosphite esters photoactivatable group varies; see definitionvaries; see definition quadricyclanes π-electrophile (e.g., norborneneNi bis(dithiolene)) cycloaddition product silyl halides alcohols silylethers sulfonate esters amines/anilines alkyl amines sulfonate esterscarboxylic acids esters sulfonate esters thiols thioethers sulfonateesters alcohols/phenols ethers sulfonyl halides amines/anilinessulfonamides 1,2,4,5-tetrazine alkene dihydropyradazine vinyl sulfonylthiols thioethers vinyl sulfonyl activated diene cyclohexenyl (Diels-Alder) *Activated esters, as understood in the art, generally have theformula —C(O)OM, where —OM is a leaving group (e.g. succinimidyloxy(—OC₄H₄NO₂), sulfosuccinimidyloxy (—OC₄H₃NO₂SO₃H), -1-oxybenzotriazolyl(—OC₆H₄N₃); 4-sulfo-2,3,5,6-tetrafluorophenyl; or an aryloxy group oraryloxy substituted one or more times by electron withdrawingsubstituents such as nitro, fluoro, chloro, cyano, or trifluoromethyl,or combinations thereof, used to form activated aryl esters; or —C(O)OMis a carboxylic acid activated by a carbodiimide to form an anhydride ormixed anhydride —C(O)OC(O)R^(a) or —C(O)OC(NR^(a))NHR^(b), wherein R^(a)and R^(b) are members independently selected from the group consistingof C₁-C₆ alkyl, C₁-C₆ perfluoroalkyl, C₁-C₆ alkoxy, cyclohexyl,3-dimethylaminopropyl, or N-morpholinoethyl). **Acyl azides can alsorearrange to isocyanates.

Some methods of forming linking groups include those taught in Slettenand Bertozzi, J. Am. Chem. Soc. electronic publication atdx.doi.org/10.1021/ja2072934; Devaraj and Weissleder, Acc. Chem. Res.electronic publication at dx.doi.org/10.1021/ar200037t; Krishnamoorthyand Begley, J. Am. Chem. Soc. electronic publication atdx.doi.org/10.1021/ja1034107; and the like.

When linking a compound of Formula I having a carboxylic acid with anamine-containing ligand or biomolecule, the carboxylic acid can first beconverted to a more reactive form, e.g, a N-hydroxy succinimide (NHS)ester or a mixed anhydride, by means of an activating reagent. Theamine-containing ligand or biomolecule is treated with the resultingactivated acyl to form an amide linkage. In a more preferred embodiment,this reaction is carried out in aqueous buffer at pH 8 to 9 with DMSO orDMF as an optional co-solvent. Alternatively, this reaction is carriedout in distilled water or in an aqueous buffer solution.

Similarly, the attachment of an isocyanate- or isothiocyanate-containingcompound of Formula I is analogous to the procedure for the carboxy dye,but no activation step is required. The amine-containing ligand orbiomolecule is treated directly with the activated acyl compound to forma urea or a thiourea linkage. In a more preferred embodiment, thereaction is carried out in aqueous buffer at pH 9 to 10 with DMSO or DMFas an optional co-solvent. Alternatively, this reaction is carried outin distilled water or in an aqueous buffer solution.

If the compound of Formula I or biomolecule has a reactive hydroxylgroup, it can be linked to a ligand or biomolecule by means ofphosphoramidite chemistry, which ultimately forms a phosphate linkagebetween the dye and the biomolecule. For examples of such labelingmethods, see U.S. Pat. No. 6,027,709, which discloses many preferredlinking groups, linking methods, and biomolecules that can be readilylabeled. In one embodiment, solid-phase synthesis is preferred, asdisclosed in U.S. Pat. No. 6,027,709.

In a preferred embodiment, the biomolecule is DNA or RNA. Use ofphosphoramidite chemistry allows labeling of a DNA or an RNA during thesynthesis process. The protected nucleotide is labeled while attached toa solid-phase support. The free 5′-OH group is reacted with thephosphoramidite and a tetrazole activator to form a phosphite linkagewhich subsequently is oxidized to phosphate. The labeled DNA or RNA isthen cleaved from the solid phase by means of ammonia or by anotherstandard procedure.

It is generally preferred to prepare a phosphoramidite of a cyanine dyeto label DNA molecules in a DNA synthesizer. It is also preferred toattach the dye to the 5′ end of a protected, support-bondedoligonucleotide through standard phosphoramidite chemistry. For a listof preferred label terminators for use in DNA sequencing, see U.S. Pat.No. 5,332,666.

In another preferred embodiment, the biomolecule is an antibody. It ispreferred that antibody labeling is carried out in a buffer optionallyincluding an organic co-solvent, under basic pH conditions, and at roomtemperature. It is also preferred that the labeled antibody be purifiedby dialysis or by gel permeation chromatography using equipment such asa SEPHADEX® G-50 column to remove any unconjugated compound of FormulaI. Those of skill in the art will know of other ways and means forpurification.

In still another preferred embodiment, the biomolecule contains a thiolgroup that forms the linking group by reaction with a maleimidylsubstituent at R¹⁶. In a more preferred embodiment, the biomolecule is aprotein, a peptide, an antibody, a thiolated nucleotide, or a thiolateddeoxynucleotide.

In yet other aspects, the linking group or biomolecule comprises apolymer. In a preferred embodiment, the polymer is a member selectedfrom the group of a PEG, a copolymer of PEG-polyurethane, and acopolymer of PEG-polypropylene. In still yet other aspects, the linkinggroup is a member selected from the group of a polysaccharide, apolypeptide, an oligosaccharide, a polymer, a co-polymer and anoligonucleotide.

In one aspect, biomolecules can be labeled according to the presentinvention by means of a kit. In certain instances, the kit comprises abuffer and a dye as disclosed in the instant application (i.e., acompound of Formula I or Formula Ia). Preferably, the kit contains acoupling buffer such as 1 M KH₂PO₄ (pH 5), optionally with added acid orbase to modify the pH (e.g., pH 8.5 is preferred for reactions withsuccinimide esters and pH 7 is preferred for reactions with maleimides).Preferably, the buffer has a qualified low fluorescence background.

Optionally, the kit can contain a purification sub-kit. After labeling abiomolecule with a preferred dye, the labeled biomolecule may beseparated from any side reaction products and any free hydrolyzedproduct resulting from normal hydrolysis. For biomolecules containing 13or fewer amino acids, preparative thin layer chromatography (TLC) canremove impurities. In certain instances, preparative TLC, optionallyperformed with commercially available TLC kits, can be used to purifydye-labeled peptides or proteins.

For larger biomolecules such as larger peptides or proteins, a SEPHADEX®G-15, G-25, or G-50 resin may remove unwanted derivatives. In certaininstances, a Gel Filtration of Proteins Kit, which is commerciallyavailable from Life Sciences, can be used to separate dye-labeledpeptides and proteins from free dye. The labeled biomolecules thatremain after desalting can often be used successfully without furtherpurification. In some cases, it may be necessary to resolve and assessthe activity of the different products by means of HPLC or otherchromatographic techniques.

Bioconjugate Compounds

In another embodiment of the invention, a bioconjugate of the Formula IIis provided:

wherein Q^(L) is a three-polymethine-carbon segment:

wherein the segment is the central portion of a seven-polymethine-carbonpolymethine bridge.

Q^(L), R¹, R^(2a), R^(2b), R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹²,R¹³, R¹⁴, R¹⁵, L, and Y are as previously defined for the compounds ofFormulas I, Ia, and Ib respectively, including all preferred embodimentsthat are identified herein.

Each Z is independently selected from the group consisting of -L-R¹³ and-L-R^(L). In a more preferred aspect, Z is -L-R^(L), wherein L is abond.

In still another alternative preferred aspect, the Z group's L group isa bond, and R¹³ or R^(L) is connected directly to -L-Y— or directlybonded to the phenyl ring itself if L and Y are also bonds.

Each R^(L) comprises 1) a linking group that connects the cyanine dyecompound to a biomolecule; and 2) the biomolecule to which it isconnected (i.e., the linking group and the biomolecule connectedthereby), wherein the compound comprises at least one R^(L). Preferredlinking groups are indicated in Table 1 (column C). In a particularlypreferred aspect, the linking group is an amide or an ester. In a moreparticularly preferred aspect, the linking group is an amide.

The compound has a balanced charge. In a preferred aspect, thecompound's net anionic charge is balanced by alkali metal counterions(e.g., sodium or potassium). In a more preferred aspect, at least one ofthe counterions is sodium. Alternatively, all of the counterions aresodium.

In another preferred embodiment of the bioconjugate, any preferredembodiments or aspects of the inventive compound of Formulas I, Ia, orIb can included in the embodiment of a bioconjugate. Representativeexamples of preferred compounds of Formulas I, Ia, or Ib that correspondto preferred bioconjugate embodiments are described in the dependentclaims of the instant application.

In certain aspects, a preferred biomolecule for the instant invention isselected from the group containing an acyclo terminator triphosphate, anantibody, an antigen, an avidin, a carbohydrate, a deoxy nucleic acid, adideoxy nucleotide triphosphate, an enzyme cofactor, an enzymesubstrate, a fragment of DNA, a fragment of RNA, a hapten, a hormone, anucleic acid, a nucleotide, a nucleotide triphosphate, a nucleotidephosphate, a nucleotide polyphosphate, an oligosaccharide, a peptide,PNA, a polysaccharide, a protein, a streptavidin, and the like.

Suitable nucleotides include nucleoside polyphosphates, including, butnot limited to, deoxyribonucleoside polyphosphates, ribonucleosidepolyphosphates, dideoxynucleoside polyphosphates, carbocyclic nucleosidepolyphosphates and acyclic nucleoside polyphosphates and analogsthereof. Suitable nucleotides also include ucleotides containing 3, 4,5, 6, or more phosphate groups, in the polyphosphate chain, where thephosphate (e.g., α, β, γ, ε, or terminal phosphate), sugar, base, orcombination thereof is labeled with a compound of Formula I. Thepolyphosphate nuceotides include, but are not limited to,tetraphosphates, pentaphosphates, hexaphosphates, heptaphosphates, andthe like. The bases include for example, purines, (adenine and guanine)pyrimidines, (thymine, uracil and cytosine) and derivatives thereof.

In certain instances, the dye of Formula I is attached to the phosphate(e.g. α, β, γ, ε-phosphate or terminal phosphate) through aphosphorothioate linkage (see, for example, U.S. Pat. No. 6,323,186,incorporated herein by reference), heteroatom, or functional group A, orB, resulting in linkage C of Table 1. See also U.S. Pat. No. 6,399,335(incorporated herein by reference) entitled “γ-phosphoester nucleosidetriphosphates,” which provides methods and compositions for polymerizingparticular nucleotides with a polymerase using γ-phosphoester linkednucleoside triphosphates. Other ways of linking the compounds of FormulaI to a nucleotide are known to those of skill in the art. Using thesenucleotides with a DNA polymerase can lead to identification of specificnucleotides in a DNA or RNA sequence by identification of the labeledpyrophosphate or polyphosphate released upon incorporation of thenucleotide base into RNA or DNA. (See for example, U.S. Pat. No.6,232,075, US Pub. No. 2004/0241716 and U.S. Pat. No. 7,452,698 each ofwhich is incorporated herein by reference).

More preferred aspects include an antibody, an avidin, and astreptavidin. Even more preferred aspects include a goat anti-mouse(GAM) antibody, a goat anti-rabbit (GAR) antibody, and streptavidin.

In certain other aspects, preferred biomolecules for the instantinvention include somatostatin, endostatin, a carbohydrate, anoligosaccharide, an aptamer, a liposome, PEG, an angiopoietin,angiostatin, angiotensin II, a₂-antiplasmin, annexin V, β-cyclodextrintetradecasulfate, endoglin, endosialin, endostatin, epidermal growthfactor, fibrin, fibrinopeptide p, fibroblast growth factor, FGF-3, basicfibronectin, fumagillin, heparin, hepatocycle growth factor, hyaluronan,aninsulin-like growth factor, an interferon-α, β inhibitor, ILinhibitor, laminin, leukemia inhibitory factor, linomide, ametalloproteinase, a metalloproteinase inhibitor, an antibody, anantibody fragment, an acyclic RGD peptide, a cyclic RGD peptide,placental growth factor, placental proliferin-related protein,plasminogen, plasminogen activator, plasminogen activator inhibitor-1, aplatelet activating factor antagonist, platelet-derived growth factor, aplatelet-derived growth factor receptor, a platelet-derived growthfactor receptor, platelet-derived endothelial cell growth factor,pleiotropin, proliferin, proliferin-related protein, a selectin, SPARC,a snake venom, substance P, suramin, a tissue inhibitor of ametalloproteinase, thalidomide, thrombin, thrombin-receptor-activatingtetradecapeptide, transformin growth factor-α, β, transforming growthfactor receptor, tumor growth factor-α, tumor necrosis factor,vitronectin, and the like.

In still other aspects, preferred biomolecules include a carbohydrateand a carbohydrate derivative. Representative examples includeglucosamine, a glyceraldehyde, erythrose, threose, ribose, arabinose,xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose,galactose, talose, erythrulose, ribulose, xylulose, psicose, fructose,sorbose, tagatose, and a derivative thereof. Even more preferredbiomolecules include 2-deoxy-D-glucose, 2-deoxy-L-glucose, and racemic2-deoxyglucose.

In yet still other aspects, the biomolecule can be a ligand that hasaffinity for a receptor selected from the group of EGFR, Her2, PDGFR,IGFR, c-Ryk, c-Kit, CD24, integrins, FGFR, KFGR, VEGFR, TRAIL decoyreceptors, retinoid receptor, growth receptor, PPAR, vitamin receptor,glucocordicosteroid receptor, Retinoid-X receptor, RHAMM, high affinityfolate receptors, Met receptor, estrogen receptor and Ki67.

Alternatively, the biomolecule is selected from the group ofsomatostatin, endostatin, a carbohydrate, a monosaccaride, adisaccharide, a trisaccharide, an oligosaccharide, aptamer, liposome andpolyethylene glycol.

In yet another aspect, the biomolecule is a small-molecule drug ordrug-like molecule such as a tetracycline antibiotic, a tetracyclinederivative, and calcein.

Alternatively, the biomolecule is a small-molecule drug or peptide.

In other aspects, a cyanine dye set for the in the present invention isconjugated to a biological cell. Preferably, the dye is conjugated bymeans of an R^(L) linking group.

In other aspects, a preferred biomolecule for the instant invention isselected from the group containing an antigen and a hapten. Preferably,the biomolecule is an immunogen.

In other aspects, a preferred biomolecule for the instant invention isselected from the group containing an enzyme cofactor and an enzymesubstrate.

In other aspects, a preferred biomolecule for the instant invention isselected from the group containing an amino acid, a carbohydrate, ahapten, a hormone, a glycoprotein, a liposome, a nucleic acid, anucleotide, a nucleotide triphosphate, a nucleotide polyphosphate, anoligosaccharide, a peptide, a peptide nucleic acid, a polyalkyleneglycol, a polysaccharide, a protein, a small-molecule drug, and a snakevenom.

More preferably, the preferred biomolecule is selected from the groupcontaining angiostatin, endostatin, fumagillin, a fumagillin derivative,placental proliferin-related protein, plasminogen, somatostatin, andthalidomide.

Alternatively, the biomolecule is an aptamer.

Alternatively, the biomolecules is selected from the group containing anantibody and an antibody fragment.

Alternatively, the biomolecule is selected from the group containingpolyethylene glycol.

Alternatively, the biomolecule is selected from the group containing anangiopoietin, epidermal growth factor, a fibroblast growth factor,hepatocyte growth factor, an insulin-like growth factor, placentalgrowth factor, platelet-derived growth factor, a platelet-derived growthfactor receptor, a platelet-derived endothelial cell growth factor,transforming growth factor-α, transforming growth factor-13, andtransforming growth factor receptor. More preferably, the fibroblastgrowth factor is fibroblast growth factor 3.

Alternatively, the biomolecule is selected from the group containing anacyclic RGD peptide, a cyclic RGD peptide, and endosialin. Preferably,the biomolecule is an acyclic RGD peptide, a cyclic RGD peptide, or aderivative thereof. More preferably, the cyclic RGD peptide is cyclo(Arg-Gly-Asp-D-Phe-Lys) (i.e., c(RGDfK)).

Alternatively, the biomolecule is selected from the group containingα₂-antiplasmin, plasminogen, plasminogen activator, plasminogenactivator inhibitor-1, and plasminogen activator inhibitor-2.

Alternatively, the biomolecule is selected from the group containingfibrin, fibrinopeptide β, thrombin, and thrombin-receptor-activatingtetradecapeptide.

Alternatively, the biomolecule is selected from the group containing anacyclo terminator triphosphate, a deoxynucleic acid, a ribonucleic acid,a a nucleotide, a nucleotide triphosphate, a nucleotide polyphosphate,and a peptide nucleic acid.

Alternatively, the biomolecule is selected from the group containing afragment of RNA and a fragment of DNA.

Alternatively, the biomolecule is selected from the group containingangiotensin II and substance P.

Alternatively, the biomolecule is selected from the group containing alectin and a selectin.

Alternatively, the biomolecule is selected from the group containingendoglin, a laminin, a fibronectin, SPARC, and vitronectin.

Alternatively, the biomolecule is selected from the group containing ametalloproteinase and a metalloproteinase inhibitor.

Alternatively, the biomolecule is a tissue inhibitor of ametalloproteinase.

Alternatively, the biomolecule is a platelet activating factorantagonist.

Alternatively, the biomolecule is selected from the group containingβ-cyclodextrin tetradecasulfate, heparin, and hyaluronan.

Alternatively, the biomolecule is an annexin.

Alternatively, the biomolecule is selected from the group containinginterleukin inhibitor, leukemia inhibitory factor, pleiotropin, andtumor necrosis factor. More preferably, the biomolecule is aninterleukin-1 receptor antagonist.

Alternatively, the biomolecule is selected from the group containingproliferin and a proliferin-related protein.

Alternatively, the biomolecule is selected from the group containingcalcein, laquinimod, linomide, and suramin.

Alternatively, the biomolecule is an interferon-α,β inhibitor.

Alternatively, the biomolecule is selected from the group containingtyramine and a tyramine derivative.

Alternatively, the biomolecule is selected from the group containing anavidin, biotin, and a streptavidin.

Alternatively, the biomolecule is selected from the group containing aglucosamine, a glyceraldehyde, erythrose, threose, ribose, arabinose,xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose,galactose, talose, erythrulose, ribulose, xylulose, psicose, fructose,sorbose, tagatose, and a derivative thereof. More preferably, thebiomolecules include 2-deoxy-D-glucose, 2-deoxy-L-glucose, and racemic2-deoxyglucose.

Methods of Imaging

In another embodiment, the compounds of Formula I, Ia, or Ib can be usedas in vitro or in vivo optical imaging agents of tissues and organs invarious biomedical applications. In one embodiment, the presentinvention provides a method for imaging, the method comprisingadministering a compound of Formula I, Ia, or Ib.

In certain preferred aspects of the invention, any of the embodiments oraspects of the inventive compound of Formula I, Ia, or Ib that aredescribed herein can be used in the method of imaging. Representativeexamples of preferred compounds for use in the method are described inthe specification and the dependent claims of the instant application.

In another embodiment, the present invention provides a method forimaging, the method comprising administering a compound of Formula II,IIa, or lib.

In certain preferred aspects of the invention, any of the embodiments oraspects of the inventive compound of Formula II that are describedherein can be used in the method of imaging. Representative examples ofpreferred compounds for use in the method are described in thespecification and the dependent claims of the instant application.

In certain preferred aspects, the compounds of the present invention areused as in vivo imaging agents of tissues and organs in variousbiomedical applications including, but not limited to, tomographicimaging of organs, monitoring of organ functions, coronary angiography,fluorescence endoscopy, imaging of tumors, laser guided surgery,photoacoustic and sonofluorescence methods, and the like. In one aspect,the compounds of the invention are useful for the detection of thepresence of tumors and other abnormalities by monitoring the bloodclearance profile of the dyes. In another aspect of the invention, thecompounds are useful for laser assisted guided surgery for the detectionof micro-metastases of tumors upon laparoscopy. In yet another aspect,the compounds are useful in the diagnosis of atherosclerotic plaques andblood clots.

In further aspects, the compounds of the present invention are used inthe imaging of: (1) ocular diseases in ophthalmology, for example, toenhance visualization of chorioretinal diseases, such as vasculardisorders, retinopathies, neovascularization, and tumors via directmicroscopic imaging; (2) skin diseases such as skin tumors via directmicroscopic imaging; (3) gastrointestinal, oral, bronchial, cervical,and urinary diseases and tumors via endoscopy; (4) atheroscleroticplaques and other vascular abnormalities via flexible endoscopiccatheters; (5) breast tumors via 2D- or 3D-image reconstruction; and (6)brain tumors, perfusion, and stroke via 2D- or 3D-image reconstruction.

In certain aspects, the compounds of the invention that arebioconjugates are particularly useful for imaging tumors, tissues, andorgans in a subject. For example, the existence of cancer cells orcancer tissues can be verified by labeling an anti-tumor antibody with acompound of Formula I and then administering the bioconjugated antibodyto the subject for detection and imaging of the tumor. Conjugatesbetween the dye compound and other antibodies, peptides, polypeptides,proteins, ligands for cell surface receptors, small molecules, and thelike are also useful agents for the in vivo imaging of tumors, tissues,and organs in a subject.

In certain aspects, the compounds of the invention may be administeredeither systemically or locally to the organ or tissue to be imaged,prior to the imaging procedure. In one aspect, the compounds areadministered intravenously. In another aspect, the compounds areadministered parenterally. In yet another aspect, the compounds areadministered enterally. The compositions used for administration of thecompound typically contain an effective amount of the compound orconjugate along with conventional pharmaceutical carriers and excipientsappropriate for the type of administration contemplated. For example,parenteral formulations advantageously contain a sterile aqueoussolution or suspension of a compound of Formula I, Ia, or Ib; or abioconjugate of Formula II, IIa, or IIb. Compositions for enteraladministration typically contain an effective amount of the compound orbioconjugate in aqueous solution or suspension that may optionallyinclude buffers, surfactants, thixotropic agents, flavoring agents, andthe like.

In certain aspects, the compositions are administered in doses effectiveto achieve the desired optical image of a tumor, tissue, or organ. Suchdoses may vary widely, depending upon the particular compound orbioconjugate employed, the tumor, tissue, or organ subjected to theimaging procedure, the imaging equipment being used, and the like.

In certain aspects, the method of the present invention provides foradministering to the subject a therapeutically effective amount of acompound; a targeting agent, such as a bioconjugate; or mixturesthereof. In one aspect, the targeting agent selectively binds to thetarget tissue. Light at a wavelength or waveband corresponding to thatwhich is absorbed by the photosensitizing agent is then administered. Inanother aspect, the compounds of the present invention act agentscapable of binding to one or more types of target cells or tissues, whenexposed to light of an appropriate waveband, absorb the light, causingsubstances to be produced that illuminate, impair or destroy the targetcells or tissues. Preferably, the compound is nontoxic to the subject towhich it is administered or is capable of being formulated in a nontoxiccomposition that can be administered to the subject. In addition,following exposure to light, the compound in any resulting photodegradedform is also preferably nontoxic.

In yet another aspect, the compounds of the present invention areadministered by any means known in the art, including, but not limitedto, ingestion, injection, transcutaneous administration, transdermaladministration, and the like. Preferably, the compounds are administeredtranscutaneously to a subject.

In certain aspects, during imaging, the light passes through unbrokentissue. Where the tissue layer is skin or dermis, such transcutaneousimaging includes transdermal imaging, and it will be understood that thelight source is external to the outer skin layer. In some aspects (i.e.,transillumination), the light passes through a tissue layer, such as theouter surface layer of an organ (e.g., the liver). In such cases, thelight source is preferably external to the organ, but internal orimplanted within the subject or patient.

In further aspects of the invention, the target tumor, tissue, or organfor treatment is selected from the group of vascular endothelial tissue,an abnormal vascular wall of a tumor, a solid tumor, a tumor of thehead, a tumor of the neck, a tumor of a the gastrointestinal tract, atumor of the liver, a tumor of the breast, a tumor of the prostate, atumor of the ovary, a tumor of the uterus, a tumor of the testicle, atumor of the lung, a nonsolid tumor, malignant cells of one of ahematopoietic tissue and a lymphoid tissue, lesions in the vascularsystem, a diseased bone marrow, neuronal tissue or diseased neuronaltissue, and diseased cells in which the disease is one of an autoimmuneand an inflammatory disease. In yet a further aspect, the target tissueis a lesion in the vascular system of a type selected from the group ofatherosclerotic lesions, arteriovenous malformations, aneurysms, andvenous lesions.

In still further aspects, the forms of energy include, but are notlimited to, light (i.e., radiation), thermal, sonic, ultrasonic,chemical, light, microwave, ionizing (such as x-ray and gamma ray),mechanical, and electrical. The term “radiation” as used herein includesall wavelengths and wavebands. Preferably, the radiation wavelength orwaveband is selected to correspond with or at least overlap thewavelengths or wavebands that excite the photosensitizing agent.Compounds of the instant invention typically have one or more absorptionwavebands that excite them to produce the substances which illuminate,damage or destroy target cells, tissues, organs, or tumors. Preferably,the radiation wavelength or waveband matches the excitation wavelengthor waveband of the photosensitizing agent and has low absorption by thenon-target cells and the rest of the subject, including blood proteins.More preferably, the radiation wavelength or waveband is within the NIRrange of about 600 nm to about 1000 nm or a related range thereof (e.g.,the ranges that are described in the instant claims).

In certain aspects, the compounds of the present invention are used todirectly stain or label a sample so that the sample can be identified orquantitated. For instance, such compounds can be added as part of anassay for a biological target analyte, as a detectable tracer element ina biological or non-biological fluid; or for such purposes asphotodynamic therapy of tumors, in which a dyed sample is irradiated toselectively destroy tumor cells and tissues; or to photoablate arterialplaque or cells, usually through the photosensitized production ofsinglet oxygen.

Typically, the sample is obtained directly from a liquid source or as awash from a solid material (organic or inorganic) or a growth medium inwhich cells have been introduced for culturing, or a buffer solution inwhich cells have been placed for evaluation. Where the sample comprisescells, the cells are optionally single cells, including microorganisms,or multiple cells associated with other cells in two or threedimensional layers, including multicellular organisms, embryos, tissues,biopsies, filaments, biofilms, and the like.

A detectable optical response as used herein includes a change in, oroccurrence of, an optical signal that is detectable either byobservation or instrumentally. Typically the detectable response is achange in fluorescence, such as a change in the intensity, excitation oremission wavelength distribution of fluorescence, fluorescence lifetime,fluorescence polarization, or a combination thereof. The degree and/orlocation of staining, compared with a standard or expected response,indicates whether and to what degree the sample possesses a givencharacteristic. Some compounds of the invention may exhibit littlefluorescence emission, but are still useful as chromophoric dyes. Suchchromophores are useful as energy donors in fluorescence (or Förster)resonance energy transfer (FRET) applications, or to impart the desiredcolor to a sample or portion of a sample.

FRET is a process by which a donor molecule (e.g., a dye) absorbs light,entering an excited state. Rather than emitting light, the firstmolecule transfers its excited state to an acceptor molecule with otherproperties (e.g., a dye fluorescing at a different wavelength or aquencher), and the acceptor fluoresces or quenches the excitation.Because the efficiency of the transfer is dependant on the twomolecules' proximity, it can indicate information about molecularcomplex formation or biomolecular structure. It can also indicate wherea particular complex is located within a cell or organism (e.g., FREToptical microscopy). For ways to use similar dyes as acceptors(quenchers) in FRET processes, see X. Peng, H. Chen, D. R. Draney, W.Volcheck, A. Schultz-Geschwender, and D. M. Olive, “A nonfluorescent,broad-range quencher dye for Förster resonance energy transfer assays,”Anal. Biochem 2009, 388(2): 220-228.

In certain aspects, a suitable FRET acceptor is disclosed as in WO2007/005222, which is incorporated herein by reference. The compoundsinclude the following essentially non-fluorescent cyanine dyes offormula III:

wherein the substituents in formula III are defined as follows: R¹ andR² are each independently selected from the group consisting of hydrogenand optionally substituted (C₁-C₆)alkyl. Alternatively, R¹ and R²together with the

group to which they are bonded form a 5- to 7-membered ring, the ringbeing optionally substituted with 1 to 4 substituents selected from thegroup consisting of halogen, cyano, sulfonate, (C₁-C₈)haloalkyl,hydroxy, (C₁-C₆)alkoxy and optionally substituted (C₁-C₈)alkyl.

In Formula III, R³ and R⁴ are each independently an optionallysubstituted (C₁-C₆)alkyl, and may optionally join together with theatoms to which they are attached to form a 5- to 7-membered carbocyclicring; or alternatively, the substituents R³ and R⁴ are replaced with thegroup

wherein B is (C₁-C₆)alkyl; or B and R^(9a) together with the carbonatoms to which they are attached join to form a 5- or 6-membered ringoptionally having 1 or 2 heteroatoms and optionally having up to 3double bonds.

In Formula III, the substituents R⁵ and R⁶ are each independently anoptionally substituted (C₁-C₆)alkyl, and may optionally join togetherwith the atom to which they are attached to form a ring.

In Formula III, the substituents R⁷ and R⁸ are each independentlyselected from the group consisting of optionally substituted(C₁-C₆)alkyl, optionally substituted aryl(C₁-C₆)alkyl, optionallysubstituted heteroaryl(C₁-C₆)alkyl, —(CH₂)_(c)R¹³ and —(CH₂)_(d)R¹⁵.Indices c and d are each independently an integer from 1-50. R¹³ is afunctional group that does not directly react with a carboxyl, hydroxyl,amino or thio group on a biomolecule. R¹⁵ is a linking group selectedfrom the group consisting of mercapto, amino, haloalkyl,phosphoramidityl, N-hydroxy succinimidyl ester, sulfoN-hydroxysuccinimidyl ester, isothiocyanato, iodoacetamidyl, maleimidyland an activated carboxylic acid.

In Formula III, the substituents R^(9a-9d) and R^(10a-10d) are eachindependently selected from the group consisting of hydrogen, optionallysubstituted (C₁-C₆)alkyl, —SO₃Cat⁺, halogen, —C(O)OR¹¹, —C(O)NR¹¹R¹²,—C(O)O(CH₂)_(d)R¹⁵, —C(O)NR¹¹(CH₂)_(d)R¹⁵, —NR¹²C(O)O(CH₂)_(d)R¹⁵,—NR¹²C(O)OR¹¹, —(CH₂)_(d)R¹⁵, —S(O)₂NR¹²(CH₂)_(d)R¹⁵, —R¹⁵ and —NR²⁰R²¹,wherein Cat⁺ is a cation. The substituents R¹¹ and R¹² are eachindependently selected from the group consisting of hydrogen and(C₁-C₆)alkyl; R²⁰ and R²¹ are each independently selected from the groupconsisting of hydrogen, optionally substituted (C₁-C₈)alkyl,CatO₃S(C₁-C₅₀)alkylene.

In Formula III, alternatively, any two substituents of R^(10a-10d)located on adjacent atoms, together with the atoms to which they areattached, join to form a 5- or 6-membered ring optionally having 1 or 2heteroatoms and optionally having up to 3 double bonds; wherein the ringmay be further substituted with 1 to 3 substituents selected from thegroup consisting of optionally substituted (C₁-C₆)alkyl, —SO₃ ⁻Cat,halogen, —C(O)OR¹¹, —C(O)NR¹¹R¹², C(O)O(CH₂)_(d)R¹⁵,—C(O)NR¹¹(CH₂)_(d)R¹⁵, —NR¹²C(O)O(CH₂)_(d)R¹⁵, —NR¹²C(O)OR¹¹,—S(O)₂NR¹²(CH₂)_(d)R¹⁵, —R¹⁵ and —NR²⁰R²¹

In Formula III, the variable a is an integer from 0-3 and the variable bis an integer from 0-2. A is selected from the group consisting ofhydrogen, halogen, cyano, optionally substituted (C₁-C₈)alkyl,optionally substituted (C₁-C₆)dialkylamino, optionally substitutedalkylthio, —(CH₂)_(d)R¹⁵, —R¹⁵, optionally substituted(C₁-C₆)heteroalkyl, phenoxy and an optionally substituted aryloxy grouphaving the formula

wherein R^(36a)—R^(36e) are each independently selected from the groupconsisting of hydrogen, —SO₃Cat⁺, —(CH₂)_(d)R¹⁵, —C(O)O(CH₂)_(d)R¹⁵,—C(O)NR¹¹(CH₂)_(d)R¹⁵, —NR¹²C(O)O(CH₂)_(d)R¹⁵, —S(O)₂NR¹²(CH₂)_(d)R¹⁵,—R¹⁵, (C₁-C₆)alkyl, carboxyl and NR²⁰R²¹.

The compounds of Formula III have at least one linking group. In certainaspects, the compounds of the invention have one or more linking groupssuch as for example, 1, 2, 3 or more linking groups. The at least onelinking group R¹⁵ can be attached at various positions on the compoundof Formula III.

In certain aspects, for biological applications, the compounds of theinvention are typically used in an aqueous, mostly aqueous oraqueous-miscible solution prepared according to methods generally knownin the art. The exact concentration of compound is dependent upon theexperimental conditions and the desired results, but ranges of 0.00001mM up to 0.1 mM, such as about 0.001 mM to about 0.01 mM, are possible.The optimal concentration is determined by systematic variation untilsatisfactory results with minimal background fluorescence isaccomplished.

In certain aspects, the method may involve treatment of an animal orsample with a dose comprising a compound of Formula I, a bioconjugate ofFormula II, a bioconjugate of Formula III, or any of the aspects orembodiments thereof. The exact concentration of compound is dependentupon the subject and the desired results. In certain embodiments, a doseof at least about 0.001, 0.005, 0.01, 0.025, 0.05, or 0.075 mg/kg isused. Alternatively, a dose of at most about 0.001, 0.005, 0.01, 0.025,0.05, or 0.075 mg/kg is used. In certain other embodiments, a dose of atleast about 0.1, 0.25, 0.5, or 0.75 mg/kg is used. Alternatively, a doseof at most about 0.1, 0.25, 0.5, or 0.75 mg/kg is used. In still otherembodiments, a dose of at least about 0.1, 0.25, 0.5, or 0.75 mg/kg isused. Alternatively, a dose of at most about 0.1, 0.25, 0.5, or 0.75mg/kg is used. In yet still other embodiments, a dose of at least about1, 2.5, 5, or 7.5 mg/kg is used. Alternatively, a dose of at most about1, 2.5, 5, or 7.5 mg/kg is used. In additional other embodiments, a doseof at least about 10, 25, 50, or 75 mg/kg is used. Alternatively, a doseof at most about 10, 25, 50, or 75 mg/kg is used. In additional stillother embodiments, a dose of at least about 100, 250, 500, or 750 mg/kgis used. Alternatively, a dose of at most about 100, 250, 500, or 750mg/kg is used. Other amounts for administration of an effective dose maybe readily determined by one of skill in the art.

In certain aspects, the compounds are most advantageously used to stainsamples with biological components. The sample can compriseheterogeneous mixtures of components (e.g., mixtures including intactcells, fixed cells, cell extracts, bacteria, viruses, organelles, andcombinations thereof), or a single component or homogeneous group ofcomponents (e.g. natural or synthetic amino acid, nucleic acid orcarbohydrate polymers, or lipid membrane complexes). Within theconcentrations of use, these compounds are generally non-toxic to livingcells and other biological components.

The compound is combined with the sample in any way that facilitatescontact between the compound and the sample components of interest.Typically, the compound or a solution containing the compound is simplyadded to the sample. Certain compounds of the invention, particularlythose that are substituted by one or more sulfonic acid moieties, tendto be impermeant to membranes of biological cells, and once insideviable cells are typically well retained. Treatments that permeabilizethe plasma membrane, such as electroporation, shock treatments or highextracellular ATP, can be used to introduce selected compounds intocells. Alternatively, selected dye compounds can be physically insertedinto cells, e.g., by pressure microinjection, scrape loading, patchclamp methods, or phagocytosis.

Alternatively, dye compounds can be conjugated to a biomolecule thatincreases their uptake into cells (e.g., cell-penetrating peptides suchas Tat, penetratin, transportin, derivatives thereof (e.g., Tatderivatives incorporating β- and γ-amino acids), and the like). Thisgeneral approach is usable in vitro or in vivo.

In certain aspects, at any time after or during staining, the sample isilluminated with a wavelength of light selected to give a detectableoptical response, and observed with a means for detecting the opticalresponse. Equipment that is useful for illuminating the compounds of theinvention includes, but is not limited to, hand-held ultraviolet lamps,mercury arc lamps, xenon lamps, lasers and laser diodes. Theseillumination sources are optionally integrated into laser scanners,fluorescence microplate readers, standard or minifluorometers, orchromatographic detectors. Preferred aspects of the invention arecompounds that are excitable at or near the wavelengths 674-685 nm,685-690 nm, 690-695 nm, 690-700 nm, 700-720 nm, 720-740 nm, 740-750 nm,750-760 nm, 760-770 nm, 770-780 nm, 780 nm, 780-790 nm, 790-800 nm,800-810 nm, 810 nm, 810-820 nm, 820-830 nm, and beyond (e.g., 850 nm),as these regions closely match the output of exemplary compounds or ofrelatively inexpensive excitation sources.

The optical response is optionally detected by visual inspection, or byuse of any of the following devices: CCD cameras, video cameras,photographic film, laser-scanning devices, fluorometers, photodiodes,quantum counters, epifluorescence microscopes, scanning microscopes,flow cytometers, fluorescence microplate readers, or by means foramplifying the signal such as photomultiplier tubes. Where the sample isexamined by means of a flow cytometer, examination of the sampleoptionally includes sorting portions of the sample according to theirfluorescence response.

EXAMPLES

Below, the present invention will be described by way of examples, whichare provided for illustrative purposes only. Accordingly, they are notto be construed as limiting the scope of the present invention asdefined by the appended claims.

Example 1

A mixture of 14 g of sodium 2,3,3-trimethyl-3H-indole-5-sulfonate and 14g 1,3-propanesultone in 100 mL dicholorobenzene was heated at 110° C.for 2 h. After it cooled, the solvent was decanted. The solid was thendissolved in 100 ml of acetonitrile, and 300 ml of ethyl acetate wasadded. The resulting sticky solid was again stirred in 300 ml of ethylacetate to yield 20 g of the compound 1.

Example 2

Compound 2 was prepared analogously to compound 1 except with1,4-butanesultone as a starting material.

Example 3

A mixture of 0.54 g of sodium 2,3,3-trimethyl-3H-indole-5-sulfonate and1.32 g of 6-bromohexanoic acid was heated at 120° C. for 1 h. Ethylacetate (10 mL) was added, and the reaction mixture was heated at refluxfor 15 minutes, then cooled to room temperature. The supernatant liquidwas decanted to yield the product 3.

Example 4

Compound 4 is prepared analogously to compound 1 except with sodium3-(4-carboxybutyl)-2,3-dimethyl-3H-indole-5-sulfonate as a startingmaterial. See also the procedure of U.S. Pat. Publ. No. 2007/0232805.

Example 5

A solution dimethylformamide (20 ml) in 40 ml of methylene chloride isstirred for 45 min with phosphorus bromide (10 ml) at 4 to 5° C.Subsequently, the mixture is stirred at 5° C. for another 30 min.Cyclohexanone (10 g) is added dropwise for approximately 15 min causingthe temperature to boil. After heating at reflux for 5 h, methylenechloride is distilled off to 45° C. (internal temperature), andsubsequently the remaining volatile components are removed in vacuo. Theresidue iss discharged on 0.5 kg of ice with constant cooling at 20° C.An anline/EtOH [1:1, (v/v), 20 ml] is added dropwise. Reaction wascontinued for additional 30 min after the aniline addition, and then theyellow mixture is poured into ice-cold water/conc. HCl (10:1, 11 ml).The final malonaldehyde dianil hydrochloride salts are precipitated aslight yellow solids.

The chloro-analog to bromo-compound 5 is commercially available andserves as an alternative substrate to compound 5.

Example 6

The procedure to synthesize compound 6 was analogous to Example 5 aboveexcept that 4-cyclohexanone carboxylic acid ethyl ester was a startingmaterial.

Example 7

To a solution of compound 1 (2.0 equiv) and compound 6 (1.0 equiv) inethyl alcohol (20 ml) was added sodium acetate (4.0 equiv). The mixturewas heated at 50° C. for 2 h. Diethyl ethyl ether was added toprecipitate the crude product, which was purified by chromatography (15%methanol/water) on RP—C18 silica gel. UV: λ_(MeOH)=788 nm

The chloro analog was synthesized by an analogous procedure from thechloro equivalent for compound 6.

Example 8

The procedure to synthesize compound 8 is analogous to Example 7 aboveexcept that compound 2 (2.0 equiv) and compound 5 are startingmaterials.

The chloro analog was synthesized by an analogous procedure from thecommercially available chloro equivalent for compound 5.

Example 9

To compound 2 (1.0 equiv), compound 3 (2.0 equiv), and compound 5 (1.0equiv) in ethyl alcohol (20 ml) is added sodium acetate (4.0 equiv). Themixture is heated at 50° C. for 2 h. Diethyl ethyl ether is added toprecipitate the crude product, which is purified by chromatography (15%acetonitrile/water) on RP—C18 silica gel. UV: λ_(MeOH)=788 nm

Example 10

The procedure to synthesize compound 10 is analogous to Example 9 aboveexcept that compound 4 (2.0 equiv) is a starting material.

The chloro-equivalent to bromo-compound 10 is prepared by a similarprocedure using the chloro analog to compound 5.

Example 11

Compound 11 was prepared by combining 100 mg of the bromo dye (compound8) with 70 mg 5-(3-boronophenyl)pentanoic acid and 10 mg of Pd(PPh₃)₄.The mixture was heated at reflux with 50 mL water for 1 h under nitrogengas. The green solution was separated by HPLC using a reverse-phase C18acetonitrile/water gradient in a ratio of 15:85.

Alternatively, compound 11 was prepared by the same general procedureusing the chloro-analog to compound 8 as a starting material. Thepurified product had a λ_(MeOH)=767 nm, λ_(PBS)=757 nm, and emission at775 nm. Its absorption and emission spectra in PBS are shown in FIG. 1.

Example 12

Compound 12 was prepared analogously to compound 11 (Example 11), exceptthat 2-fluorophenylboronic acid and the chloro-analog to compound 8 wereused as starting materials. UV: λ_(MeOH)=772 nm.

Example 13

Compound 13 was prepared analogously to compound 11 (Example 11), exceptthat 2,6-difluorophenylboronic acid and the chloro-analog to compound 8were used as starting materials. UV: λ_(MeOH)=782 nm.

Example 14

Compound 14 was prepared analogously to compound 11 (Example 11), exceptthat 2,6-difluorophenylboronic acid and the chloro-analog to compound 8were used as starting materials. UV: λ_(MeOH)=785 nm.

Example 15

Compound 15 was prepared by combining 100 mg of the bromo dye (compound7) with 70 mg 2,6-dichloro phenylboronic acid and 10 mg of Pd(PPh₃)₄.The mixture was refluxed with 50 ml water for 2 h under nitrogen gas.Then 10% aqueous sulfuric acid is added, and the mixture is heated atreflux for 1 h. The resulting green solution was separated by HPLC usinga reverse-phase C18 acetonitrile/water gradient in a ratio of 15:85. Thepurified product has λ_(MeOH)=782 nm.

Example 16

Compound 16 was prepared analogously to compound 15 (Example 15), exceptthat 2,4,6-trifluoro phenylboronic acid was used as a starting material.UV: λ_(MeOH)=785 nm.

Compound 16 was also prepared by the same general procedure using thechloro-analog to compound 7 as a starting material. The purifiedproduct's absorption and emission spectra in PBS are shown in FIG. 2.

Example 17

To 55 mg of compound 16 in 1 mL of DMSO was added 34 μl of triethylamineand 21 mg of N,N′-disuccinimidyl carbonate. The mixture was stirred atroom temperature for 30 min and then precipated by diethyl ethyl etherto yield the succinimidyl ester 17 as a green solid.

Example 18

5-Aminopentanoic acid (M.W. 117, 10 mg) and compound 17 (20 mg) aremixed in 50 mM phosphate buffer (10 ml, pH 8.5) for 1 h. The mixture ispurified by preparative HPLC to afford compound 18

Example 19

Compound 19 was prepared analogously to compound 16 (Example 16), exceptthat compound 9 was used as a starting material rather than compound 7.UV: λ_(MeOH)=785 nm.

Example 20

Compound 20 is prepared analogously to compound 16 (Example 16), exceptthat compound 10 is used as a starting material. UV: λ_(MeOH)=785 nm.

Example 21

Compound 21 was prepared analogously to compound 11 (Example 11), exceptthat the chloro-analog to compound 8 and 2,6-difluoro-4-methoxyphenylboronic acid were used as starting materials. UV: λ_(MeOH)=772 nm.

Example 22

Compound 22 was prepared analogously to compound 11 (Example 11), exceptthat the chloro-analog to compound 8 and 2,6-difluoro-4-butoxyphenylboronic acid were used as starting materials. UV: λ_(MeOH)=786 nm,λ_(PBS)=779 nm.

Example 23

Compound 23 was prepared analogously to compound 11 (Example 11), exceptthat the chloro-analog to compound 8 and 2,4,6-trifluoro-3-butoxyphenylboronic acid were used as starting materials. UV: λ_(MeOH)=789 nm,λ_(PBS)=782 nm; .emission at 806 nm: E_(MeOH)=300,000, E_(PBS)=240,000.

Example 24

Compound 24 is prepared analogously to compound 11 (Example 11), exceptthat the chloro-analog to compound 8 and 2,3,5,6-tetrafluoro-4-butoxyphenylboronic acid were used as starting materials. UV: λ_(MeOH)=790 nm,λ_(PBS)=786 nm; emission at 805 nm.

Example 25

Example 25 illustrates the synthesis of a fluorescence-quenching dyesodium6-((E)-2-((E)-2-(3-((E)-2-(5-(bis(3-sulfonatopropyl)amino)-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indolium-2-yl)vinyl)-2-(2-fluorophenyl)cyclohex-2-enylidene)ethylidene)-1,1-dimethyl-7-sulfonato-1H-benzo[e]indol-3(2H)-yl)hexanoate(25a).

Compound 25a was prepared by combining 50 mg of IRDye® QC-1 Carboxylate(25b, LI-COR Biosciences), 9.8 mg of 2-fluorophenylboronic acid, 4.0 mgof Pd(PPh₃)₄ 16.4 mg of sodium acetate, 200 μL of 2-methoxyethanol, and2 mL of water. The mixture was heated at reflux for 1.5 hours under anitrogen atmosphere. The compound was purified by reverse-phase C18chromatography using acetonitrile/water yielding 50 mg of a blue-greensolid (product 25a). Absorbance: λ_(Water)=785 nm, λ_(MeOH)=777 nm.

Example 26

Compound 26 was prepared by combining 100 mg of the bromo dye (compound8) with 70 mg of 2,6-difluoro-4-(methoxycarbonyl)phenylboronic acidpinacol ester and 10 mg of Pd(PPh₃)₄. The mixture was heated at refluxwith 100 ml water for 2 h under nitrogen gas. Then 2 ml 10% aqueoussulfuric acid was added, and the mixture was refluxed for 1 h. Theresulting green solution was separated by HPLC using a reverse-phase C18acetonitrile/water gradient in a ratio of 15:85. λ_(MeOH)=782 nm.

Example 27

Compound 27 is prepared analogously to compound 17 (Example 17), exceptthat compound 26 is used as a starting material.

Example 28

Compound 28 was prepared analogously to compound 18 (Example 18), exceptthat compound 27 was used as a starting material. UV: λ_(MeOH)=782 nm.

Example 29

TABLE 2 Halo Substitution Effect on Absorption Wavelength of the CyanineDye

Compound R¹ R^(7A) R⁸ R⁹ R¹⁰ R¹¹ R¹² λ_(MeOH) 11 (CH₂)₄SO₃Na H H H H(CH₂)₄COOH H 766 12 (CH₂)₄SO₃Na H H H H H F 772 13 (CH₂)₄SO₃Na H F H H HF 782 14 (CH₂)₄SO₃Na H F H F H F 785 21 (CH₂)₄SO₃Na H F H OMe H F 783 22(CH₂)₄SO₃Na H F H H OBu F 783 23 (CH₂)₄SO₃Na H F H F OBu F 789 24(CH₂)₄SO₃Na H F F OBu F F 790 16 (CH₂)₃SO₃Na COOH F H F H F 785 15(CH₂)₃SO₃Na COOH Cl H H H Cl 782 26 (CH₂)₄SO₃Na H F H COOH H F 782 64(CH₂)₄SO₃Na H H H COOH F F 772 65 (CH₂)₄SO₃Na H H H (CH2)₂COOH F F 77663a (CH₂)₄SO₃Na COOH H H H F F 770 66 (CH₂)₄SO₃Na H H H ONH(CH₂)₅COOH FF 773

Conclusion:

Halo-substituted phenyl can cause cyanine dye absorption and emissionred shift, especially, halo at the R⁸ and R¹² positions. From theexperiments, F and Cl had the same effect.

The fact that the central ring is on the periphery of the chromophoremakes it surprising that halogenations of that ring has such asignificant effect on the absorption curves for the heptamethine dyes.The fact that modifying the ring in this way in the pentamethine dyesdoes not have the same effect also makes it surprising that it works inthe heptamethine dyes.

Example 30 Preparation of Bioconjugates of GAM Antibody with Compound 17

Compound 17 is reconstituted in water to 1 mg/ml. Goat anti-mouse (GAM)IgG (H+L) antibodies are reconstituted typically at 1 mg/ml in PBSbuffer pH 8.5. The dyes are added (at various molar excesses) to the GAMantibody samples and allowed to incubate for 2 hours at room temperaturein the dark. The conjugates are extensively dialyzed againstphosphate-buffered saline (PBS) buffer to remove the unconjugated freedye. The mole dye to mole protein ratios (D/P) are calculated asdescribed below:

${D/P} = {\left\lbrack \frac{A_{785}}{ɛ_{Dye}} \right\rbrack \div \left\lbrack \frac{A_{280} - \left( {0.07 \times A_{785}} \right)}{ɛ_{antibody}} \right\rbrack}$

-   -   ε dye=250,000 M⁻¹ cm⁻¹    -   ε antibody=228,800 M⁻¹ cm⁻¹    -   0.07 is the correction factor for the dye absorption at 280 nm

In general, if the reaction pH is too low, the amide coupling reactionwill be inefficient, and the dye to protein (D/P) ratios will be muchlower than expected. If necessary, additional equivalents of NHS estercan be used to drive the reaction to completion or to increase the D/Pratio.

Example 31 Preparation of Compound 17-Streptavidin Conjugates

Compound 17 is reconstituted in water to 1 mg/mL. Streptavidin isreconstituted typically at 10 mg/ml in PBS buffer (pH 8.5). The dyes areadded (at various molar excesses) to the streptavidin samples andallowed to incubate for 2 h at room temperature in the dark. Theconjugates are extensively dialyzed against PBS buffer to remove theunconjugated free dye. The ratio of moles of dye per mole of protein iscalculated by using the equation below:

${D/P} = {\left\lbrack \frac{A_{785}}{ɛ_{Dye}} \right\rbrack \div \left\lbrack \frac{A_{280} - \left( {0.07 \times A_{785}} \right)}{ɛ_{Streptavidin}} \right\rbrack}$

In which:

-   -   ε dye=250,000 M⁻¹ cm⁻¹    -   ε streptavidin=175,000 M⁻¹ cm⁻¹    -   0.07 is the correction factor for the dye absorption at 280 nm

In general, if the reaction pH is too low, the amide coupling reactionwill be inefficient, and the dye to protein (D/P) ratios will be muchlower than expected. If necessary, additional equivalents of NHS estercan be used to drive the reaction to completion or to increase the D/Pratio.

Example 32 Preparation of Bioconjugates of RGD with Compound 17

RGD peptide (1 mg) in 400 ul DMSO is added to two equivalents ofcompound 17 in 2 ml of pH 8.5 50 mM phosphate buffer and incubated for 3h. The mixture is purified with reverse-phase HPLC and freeze-dried toyield a compound 17-RGD conjugate.

Example 33 Preparation of Bioconjugates of Cyclo-(RGDfK) with Compound17

The bioconjugate of compound 17 is prepared analogously to Example 32,except that the cyclic pentapeptide cyclo(Arg-Gly-Asp-D-Phe-Lys) is usedas a starting material.

Example 34

Western Blot Comparison of GAM/17 with GAM/IRDye 800CW

Bioconjugates of 17 were compared to GAM/IRDye 800CW conjugates in aWestern blot.

Goat anti-Mouse (GAM) secondary antibodies were labeled at threedye/protein ratios (D/P=1.8, 2.8, 3.6) by the procedure of Example 30.

TABLE 3 Materials for Compound 17/GAM Western Blot Materials DescriptionVendor Product # Lot # Extra Info Odyssey Blocking Buffer LI-COR927-40000 Q0741 IRDye ® 800CW/GAM LI-COR 926-32210 B90608-04   1 mg/mL17/GAM (D/P = 1.8) RAS 628055A 0.75 mg/mL 17/GAM (D/P = 2.8) RAS 628055B0.90 mg/mL 17/GAM (D/P = 3.6) RAS 628055C 0.79 mg/mL 4x Protein LoadingBuffer LI-COR 928-40004 C00331-01 2-color marker LI-COR 928-40001B81205-03 Dilute 1:2; load 4 uL per well Odyssey Nitrocellulose LI-COR926-31092 T908011 C32 Lysate Santa Cruz sc-2205 C1605 10% NuPAGEBis-Tris Invitrogen 827-09427 9070271 (2 gels) Gels & 10032371 (6 gels)Pan Actin Ab-5 (ACT Neomarkers MS-1295 P1 1295P909D dilute NO5) 1:1000

Methods

The Western blots contained C32 lysates and were probed with mouseanti-actin. Jurkat lysate was run (5 μg to 78 ng) by SDS PAGE andtransferred to nitrocellulose. After removal from the transfer cassette,the gels were placed on filter paper to dry. The membranes were cut inhalf at the marker and then left to dry overnight.

The blots were blocked with Odyssey Blocking Buffer+0.2% Tween 20 (OBBT)for 1 hour. The primary antibody dilution was prepared in OdysseyBlocking buffer: 85 uL of Pan Actin antibody in 85 mL of OdysseyBlocking Buffer+0.2% Tween 20. The primary antibody solution (5 ml) wasadded to each blocked membrane, which was then incubated for 1 h at roomtemperature with rotation. The membranes were then washed with PBS with0.1% Tween 20 (PBST) four times for 5 min each wash.

The secondary antibodies were evaluated at 0.2 μg/ml and 0.2 ng/ml finalconcentrations. Each solution (20 mL) was prepared by dilution to thefinal concentration with OBBT (0.2 μg/ml, 1:5000 equivalent; 0.2 ng/ml,1:50,000 equivalent). These final concentrations compared to IRDye®800CW GAM (D/P=1.8).

Each membrane was incubated in 5 mL of its respective blot for 1 h atroom temperature with gentle rotation. The membranes were then washedwith PBST four times for 5 min each wash.

Western blots were prepared in duplicate and assessed for signalintensity and the visual limit of detection (LOD) compared to a control(IRDye® 800CW/GAM) (FIGS. 3-6). Prior to scanning, the membranes werewashed with 1×PBS (no Tween). The membranes were scanned on an Odyssey.

Results

The conjugates with 17 gave a higher intensity for the actin target evenat equivalent D/P ratios compared to IRDye 800CW. The intensity is abouttwo-fold at comparable conjugate D/P ratio and about threefold at higherD/P. The background remains very low, similar to the 800CW conjugate(FIGS. 3, 5).

The conjugates with compound 17 perform reasonably well even at very lowconcentration (0.2 ng/mL) (FIGS. 4, 6). Compared to the commercial IRDye800CW conjugate, the fluorescence intensity is about six-fold atcomparable D/P and ten-fold at the highest D/P tested. The visual limitof detection for the conjugates of 17 is also slightly better than thatfor the IRDye 800CW control, about one two-fold dilution. At the sametime the background from the membrane remained very low, and nonon-specific binding of the inventive conjugates was observed even atthe highest D/P examined. These are significant benefits for theconjugates of 17 compared to the current best available technology(IRDye 800CW conjugates).

Example 35 Evaluation of RGD/17 Conjugate as In Vitro Probe

RGD (Arg-Gly-Asp), the recognition motif used to bind the integrinreceptors, was labeled with compound 17. The characteristics of RGD/17conjugate and the analogous dye carboxylate 16 were evaluated in acell-based assay.

Immunocytochemical assays provide a tool for screening a compound forspecificity of a labeled agent. Binding assays give valuable informationon affinity of the labeled agent to the intended target. Specificity ofa labeled conjugate is demonstrated by blocking the target with anantibody or by competition with the unlabeled agent.

Procedure

Compound 17 was conjugated to the peptide RGD by the general procedureset forth in Example 32.

The cell lines U87GM and PC3MLN4 were assayed. Assays included binding,blocking, and carboxylate control. Serial dilutions of compound 16 wereprepared at concentrations of 200-6.25 nM. Blocking was accomplishedwith unlabeled RGD at concentrations of 10-0.31 μM with the addition of200 nM compound 17-RGD conjugates in all test wells. The compound 16 wasevaluated as a dye control for non-specific binding at relatively highlevels from 5-0.16 μM with U87GM cells and 1-0.002 μM with PC3 cells.

Results

Compound 17/RGD conjugates successfully bound both U87GM and PC3 cells(FIG. 7).

Compound 16 was evaluated at significantly higher levels (up to 5 μM)for an assessment of stickiness and non-specific binding of the dyecomponent alone in U87GM (FIG. 7C). Relatively high binding was notedabove 2 μM. The level of compound 16 was reduced to a high of 1 μM tovisualize the baseline/threshold for non-specific binding in PC3 cells(FIG. 7F). Relative signal intensities for 17/RGD (100 nM) were 8×higher than compound 16 (100 nM) in PC3 cells.

U87GM (FIG. 7A) and PC3 (FIG. 7D) cells exhibited a dose dependentincrease in compound 17/RGD binding when incubated with increasingconcentrations. Compound 17/RGD specificity was determined by treatingU87GM cells with increasing concentration of unlabeled RGD (FIG. 7B).Results showed significant reduction in compound 17/RGD binding. PC3cell competition (FIG. 7E) showed no significant reduction in compound17/RGD binding. An increase in compound 17/RGD addition in conjunctionwith unlabeled RGD treatments may have been excessive.

In the second round of testing with U87GM cells, concentrations ofunlabeled RGD and compound 17/RGD were increased (FIGS. 8A and 8B).Binding assay concentrations utilized 0.8-400 nM while the blockingassay used 0.06-30 μM unlabeled RGD with an addition of 200 nM 17/RGD.FIG. 8 shows second round results of binding and blocking assays inU87GM cells.

A comparison of compound 17/RGD to IRDye® 800CW RGD binding was included(FIG. 8A). The two dye-labeled probes were similar in their responsewhen using U87GM cells. The effect of the blocking agent demonstratesthe inhibition of binding (FIG. 8B).

Compound 16 vs IRDye 800CW carboxylate in vivo:

Two nude mice were injected with 1 nmole of either compound 16 or IRDye800CW carboxylate. They were then imaged longitudinally over the next 24h with the Pearl Impulse imaging system. The results are presented inFIG. 9: in vivo clearance in nudes (FIG. 9A), whole body dorsal views(FIG. 9B), and the rate of clearance (FIG. 9C). The clearance data ispresented below in Table 4.

TABLE 4 Clearance Data for In Vivo Comparison of 16 and IRDye 800CWTotal Min A. Compound 16 clearance 0016459_01 800 1 109682.80 NaN 1.320.64 1 0016471_01 800 1 282046.59 NaN 3.40 1.64 17 0016483_01 800 1286573.94 NaN 3.45 1.48 45 0016495_01 800 1 266647.59 NaN 3.21 1.43 710016508_01 800 1 139736.97 NaN 1.68 0.60 190 0016520_01 800 1 76493.30NaN 0.92 0.38 276 0016535_01 800 1 1652.69 NaN 0.02 0.01 1435 B. 800CWcarboxylate clearance 0016462_01 800 1 114295.49 NaN 1.38 0.79 30016474_01 800 1 306555.97 NaN 3.69 1.85 19 0016486_01 800 1 291381.69NaN 3.51 1.85 47 0016498_01 800 1 272408.13 NaN 3.28 1.69 74 0016511_01800 1 154950.09 NaN 1.87 0.99 192 0016523_01 800 1 102698.54 NaN 1.240.66 278 0016539_01 800 1 2336.87 NaN 0.03 0.02 1439

Example 36

Example 36 illustrates the synthesis of sodium24(E)-2-((E)-3-((E)-2-(1-(1-azido-13-oxo-3,6,9-trioxa-12-azaoctadecan-18-yl)-3,3-dimethyl-5-sulfonatoindolin-2-ylidene)ethylidene)-2-(4-sulfonatophenoxy)cyclohex-1-enyl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indolium-5-sulfonate(IRDye 800CW-PEG-Azide, 29).

A solution of 11-azido-3,6,9-trioxaundecan-1-amine (Amino-PEG-Azide, 1.0mg, 4.1×10⁻³ mmol) and N,N-diisopropylethylamine (0.0020 mL, 1.1×10⁻²mmol) in anhydrous dimethyl sulfoxide (0.2 mL) was added to a reactionvessel containing IRDye 800CW NHS ester (5.0 mg, 4.3×10⁻³ mmol). Thereaction was allowed to proceed at ambient temperature for 2 hours, withperiodic agitation every 15 minutes. After HPLC analysis indicatedcomplete consumption of the IRDye 800CW NHS ester, the reaction mixturewas precipitated into anhydrous diethyl ether. The ethereal layer wasdecanted and the crude product was purified by reverse-phase HPLC.Fractions containing the desired IRDye 800CW-PEG-Azide in ≧95% purity byHPLC analysis were combined and lyophilized to afford the product 29 asa green flocculent solid (3.4 mg, 66% based on Amino-PEG-Azide). UV/Vis(methanol) λ_(max)=778 nm; LRMS (ES/water), m/z calculated for 1203.37[M+H]⁺. found 1203.6 and 602.3 [M+2H]²⁺.

Example 37

Example 37 illustrates the synthesis of a dye (IRDye800CW)/dibenzocyclooctyne substrate for click chemistry (IRDye800CW-DBCO, 30).

To a solution of IRDye 800CW NHS ester (5.0 mg, 4.3×10⁻³ mmol) andN,N-diisopropylethylamine (0.0015 mL, 8.6×10⁻³ mmol) in anhydrousdimethyl sulfoxide (0.2 mL) was added dibenzylcyclooctyne-amine(DBCO-Amine, 1.0 mg, 3.6×10⁻³ mmol). After HPLC analysis showed completeconsumption of the IRDye 800CW NHS ester, the reaction mixture wasprecipitated into anhydrous diethyl ether. The ethereal layer wasdecanted and the crude product was purified by reverse-phase HPLC.Fractions containing the desired IRDye 800CW-DBCO in ≧0.95% productpurity by HPLC analysis were combined and lyophilized to afford theproduct 30 as a flocculent green solid (3.3 mg, 68% based on DBCO-Amine)UV/Vis (methanol) λ_(max)=778 nm; LRMS (ES/water), m/z calculated for1261.4 [M+H]⁺. found 631.4 [M+2H]²⁺.

Example 38

Example 38 illustrates the synthesis of another IRDye 800CW derivative(IRDye 800CW-NH-(PEG)₂-NH-Trt, 31). See also WO 2010/002976 and Linderet al. Bioconjugate Chem. 2011, 22, 1287-1297, DOI: 10.1021/bc100457s.

A solution ofO—(N-trityl-3-aminopropyl)-O′(3-aminpropyl)-diethyleneglycol(Trt-NH-PEG₂-NH₂, 2.0 mg, 4.3×10⁻³ mmol)and N,N-diisopropylethylamine(0.002 mL, 1.1×10⁻² mmol) in anhydrous dimethyl sulfoxide (0.2 mL) wasadded to a reaction vessel containing IRDye 800CW NHS ester (5.0 mg,4.3×10⁻³ mmol). The reaction was allowed to proceed at ambienttemperature for 2 hours, with periodic vortexing at 15-minute intervals.After HPLC analysis showed complete consumption of IRDye 800CW NHSester, the reaction was precipitated into anhydrous diethyl ether. Theethereal layer was decanted and the crude product was purified by HPLC.Fractions containing the IRDye 800CW-NH-PEG₂-NH-Trt in ≧95% purity werecombined and concentrated in vacuo to afford a green film; the yield waspresumed to be quantitative.

Example 39

Example 39 illustrates the synthesis of another IRDye 800CW derivative(IRDye 800CW-NH-(PEG)₂-NH₂.TFA, 32). See also WO 2010/002976 and Linderet al. Bioconjugate Chem. 2011, 22, 1287-1297, DOI: 10.1021/bc100457s.

To a flask containing IRDye 800CW-NH-PEG₂-NH-Trt (31) (1.3 mg, 8.6×10⁻⁴)was added a solution of trifluoroacetic acid in dichloromethane(TFA/CH₂Cl₂=1:3, 5.0 mL). The dark brown reaction was briefly swirledand allowed to proceed at ambient temperature for 30 minutes. Thevolatiles were removed in vacuo and the residuals were treated againwith TFA/CH₂Cl₂ (1:3, 5.0 mL) for 30 minutes. After removing thevolatiles in vacuo, the residuals were washed with anhydrous diethylether. The ethereal layer was decanted and the IRDye800CW-NH-(PEG)₂-NH₂.TFA (32) was used without further purification; theyield was presumed to be quantitative. UV/Vis (methanol) λ_(max)=778 nm;LRMS (water) m/z calculated for 1205.4 [M+H]⁺. found 1205.6, 603.3[M+2H]²⁺.

Example 40

Example 40 illustrates the synthesis of another IRDye 800CW derivative(IRDye 800CW-PEG-Phosphine, 33a).

To a solution of IRDye 800CW-NH-(PEG)₂-NH₂.TFA (5.9 mg, 4.3×10⁻³ mmol)in anhydrous dimethyl sulfoxide (0.2 mL) was added NHS-Phosphine (2.0mg, 4.3×10⁻³ mmol, commercially available from ThermoScientific/Pierce)followed by N,N-diisopropylethylamine (0.002 mL, 1.1×10⁻² mmol). Thereaction was allowed to proceed at ambient temperature for 2 hours, withperiodic agitation at 15-minute intervals. After HPLC analysis showednear-complete consumption of the IRDye 800CW-NH-(PEG)₂-NH₂.TFA, thereaction was precipitated into anhydrous diethyl ether. The ethereallayer was decanted and the crude product was purified by HPLC. Fractionscontaining the presumed IRDye 800CW-PEG-Phosphine in ≧95% purity werecombined and concentrated in vacuo to afford a green solid (0.9 mg, 13%based on IRDye 800CW-NH-(PEG)₂-NH₂.TFA); UV-Vis (methanol) λ_(max)=778nm; LRMS (water) m/z calculated for 1551.5 [M+H]⁺. found 776.7 [M+2H]²⁺.

Example 41

Example 41 illustrates a phosphine oxide side product from the synthesisof Example 102 (IRDye 800CW-PEG-Phosphine Oxide, 33b).

This compound was isolated as a substantial byproduct from the synthesisof IRDye 800CW-PEG-Phosphine. This byproduct is nonfunctional and causesbackground problems. The compound is a green solid (17% based IRDye800CW-NH-(PEG)₂-NH₂.TFA); UV-Vis (methanol) λ_(max)=778 nm; LRMS (water)m/z calculated for 1567.5 [M+H]⁺. found 784.5 [M+2H]²⁺.

Example 42

Example 42 illustrates a non-catalyzed click chemistry synthesisreaction.

Bertozzi et al. (Aldrichimica Acta, 2010, 43(i), 15-23 and referencestherein) have developed a bio-orthogonal labeling method that employsmodified sugars. Cells incubated in a growth medium containing thesemodified azido sugars will absorb the sugars and perhaps incorporate thesugars on cell surface glycans (i.e., glycol-proteins and glycolipids).Upon exposing the azido-sugar labeled cells to appropriate phosphine oralkyne reagents, a click-type reactions will occur (either a Staudingerligation or Huisgen cycloaddition, respectively). If the phosphine oralkyne bears a reporter group (e.g., a dye), then the cells is labeledas shown below in Scheme 1:

In this example, the reaction is carried-out with IRDye 800CW as asample dye, but this proceeding is also applicable to the dye(s) of thepresent invention. The conjugate has the following advantages: (1) Thistype of bio-orthogonal labeling does not entail genetic engineering.Although many biological researchers study transgenic organisms,developmental biologists simply want to monitor “normal” changes inbiochemical morphology. (2) The click reagents are highly chemoselectiveand typically do not react with biological nucleophiles (althoughstrained alkynes may be susceptible to thiols). (3) The click chemistrycan be performed on living cells and whole organisms.

In some embodiments, the compounds of the present invention are used tomonitor azido-labeled molecules (e.g., azido sugar, protein bearingazido amino acids, lipids and site-specifically labeled proteins) inlive cells. The metabolic precursor peracetylatedN-azidoacetylmannosamine (Ac₄ManNAz) is metabolically adsorbed intocells of interest and incorporated into biomolecules that are expressedon the surface of the cells. In certain stances, the azido-sugar labeledcells are exposed to a cyclooctyne (strained alkyne) reagent conjugatedto a reporter group (e.g., dye), which generates a Huisgen cycloadditionreaction. The cyclooctyne reagent can be added to cell culture mediumand incubated with azido-sugar labeled cells at conditions that promotethe click reaction. If the azido-sugar labeled cells are in a liveorganism, the cyclooctyne reagent can be administered to the organism bymethods such as, but not limited to oral, topical and transmembraneadministration, and injection. As a result, the cyclooctyne-reporterconjugate covalently binds to the azido-sugar labeled cells, which thenlabels the cells with the reporter. In other instances, the azido-sugarlabeled cells are exposed to a phosphine reagent conjugated to areporter group (e.g., dye), which generates a Staudinger ligationbetween the phosphine and the azido sugar. This covalently binds thephosphine-reporter conjugate to the azido-sugar labeled cells, which arenow detectable using commercially available imaging systems.

In other embodiments, Ac₄ManNAz is administered to a whole organism. Incertain instances, it is injected into an animal (e.g., zebrafish,rodents, rabbits, dogs, sheep, goats, pigs, monkey, and humans;preferably zebrafish, rodents). This method delivers azides to cellsurface sialoglycoconjugates on cells found in serum and varioustissues, such as, but not limited to heart, spleen, liver, kidney,intestines and muscle. In some instances, a phosphine- oralkyne-reporter can be injected into the same animal to generate aHuisgen cycloaddition reaction or Staudinger ligation, respectively, invivo. The labeled tissues and cells can be monitored and analyzed usingwhole animal imaging systems. In other instances, tissues or cells areextracted from an Ac₄ManNAz injected animal, and then they are treatedwith a phosphine- or alkyne-reporter in vitro. Methods known to thoseskilled in the art, such as, but not limited to Western blotting, ELISA,immunocytochemistry, mass spectrometry, and high-performance liquidchromatography can then be used to detect labeled biomolecules.

Example 43

Example 43 illustrates methods of using compounds of the presentinvention to label biomolecules (e.g., proteins, lipids, carbohydrates,nucleic acids, amino acids, glycerol, fatty acids, and nucleotides) oncells as shown in Scheme 2. It also illustrates methods of in vitro andin vivo analysis of the labeled biomolecules that can serve as adetection probe. The compounds can be applied to methodologies used toinvestigate disease and therapeutic development, such as but not limitedto tumor imaging, glycan labeling, in vivo imaging, and cell surfacemodification.

Example 43 illustrates an ELISA using click chemistry.

In this example, the reaction is carried-out with IRDye 800CW as asample dye, but this proceeding is also applicable to the dye(s) of thepresent invention.

Metabolic Labeling of an Bioorthogonal Functional Group on Biomolecules

In some embodiments, an azido-labeled biomolecule is made using methodsknown to those skilled in the art. In certain instances, an unnaturalazido sugar is commercially available from a supplier (e.g.,Sigma-Aldrich). In other instances, the unnatural azido sugar, such asthe metabolic precursor peracetylated N-azidoacetylmannosamine(Ac₄ManNAz)) is synthesized according to methods known to those skilledin the art (see, e.g., Laughlin et al., Methods Enzymol, 415, 230-250(2006)). To incorporate an azido sugar into biomolecules expressed oncells cultured in vitro, the modified sugar is added to the cell culturemedia and incubated with the cells (see, e.g., Bussink et al., J. LipidRes., 48, 1417-1421 (2007); Prescher et al., Nature, 430, 873-877(2004)). Typically, Ac₄ManNAz is added to a cell culture at a finalconcentration of about 50 μM and incubated for about 3 days in cellculturing conditions. To label biomolecules expressed on cells in anorganism, an azido sugar (e.g., Ac₄ManNAz) is administered in a solutionto the organism by injection (e.g., intraperitoneal injection) at anappropriate injection schedule to ensure optimal incorporation andexpression of the modified biomolecule. See Chang et al., Proc. Natl.Acad. Sci. U.S.A., 107, 1821-1826 (2010). Non-limiting examples oforganisms include fish, rodents, rabbits, dogs, sheep, goats, pigs,monkey, and humans. Typically, Ac₄ManNAz is injected intraperitoneallyat a dose of 300 mg/kg in DMSO solution into mice once daily for 7 days.

Generation of PHOS-FLAG-Reporter Probe

Scheme 2 illustrates one embodiment of the invention, wherein aPHOS-FLAG-800CW probe is used to label specific azido-sugar labeledbiomolecules in living cells and organisms. In some embodiments, aphosphine-FLAG peptide conjugate (PHOS-FLAG; described in Laughlin etal., Methods Enzymol, 415, 230-250 (2006)) is coupled to a reportergroup (e.g., dye) using methods known to those skilled in the art. Incertain instances, the phosphine-FLAG peptide conjugate is labeled withIRDye 800CW NHS ester (LI-COR) according to manufacturer's instructions.The resulting PHOS-FLAG-800CW probe can then be covalently linked via aStaudinger ligation to an azido-labeled biomolecule expressed by a cell.

Detection of Biomolecules Labeled by Copper-Free Click Chemistry

Scheme 2 also illustrates a method of labeling a modified biomoleculefound on cell with a PHOS-FLAG-Reporter probe using “click” chemistry.

In some embodiments, a PHOS-FLAG-800CW probe is injected into an animalhaving cells that express azido-labeled biomolecules. In particularinstances, mice are injected intraperitoneally once with PHOS-FLAG-800CW(0.16 mmol/kg). Labeling of the cells with the near-infrared dye isdetected using a whole animal detection system (e.g., the Pearl Imager(LI-COR)). Dye-labeled biomolecules, tissues and cells from the animalcan be harvested from a euthanized animal and analyzed using imagers(e.g., the Odyssey System (LI-COR)).

In other embodiments, a PHOS-FLAG-800CW probe is added to an in vitrocell culture and incubated in conditions that promote a Staudingerligation reaction between the phosphine of the probe and the azide ofthe biomolecule. In certain instances, the “click” reaction is performedaccording to the following steps: 1) azido-labeled cells are collected;2) they are centrifuged at 1,500 rpm for 10 minutes, 3) they are washedthree times in cold PBS, 4) they are resuspended in 2% (v/v) fetal calfserum in PBS, 5) they are incubated with about 0.5 mM PHOS-FLAG-800CWprobe at room temperature for 3 hours under mild shaking, 6) the cellsare collected by centrifugation, and 7) they are washed three times withcold PBS. As a result of click chemistry, the labeled biomolecule iscovalently linked to a FLAG tag and near-infrared dye via the clickproduct. In some instances, the near-infrared dye-labeled cells orbiomolecules are detected and analyzed using a detection system (e.g.,Odyssey System (LI-COR)).

In certain embodiments, an ELISA-type assay is performed to detect theFLAG-tagged biomolecules expressed on cells. Protocols for ELISA-typeassays and immunocytochemistry are known to those of skill and describedin detail in reference books, such as Antibodies: A Laboratory Manual(ed. Harlow and Lane), Cold Spring Harbor Laboratory Press, New York,1988; Methods in Molecular Biology, Volume 42: ELISA, Theory andPractice (ed. Coligan et al.), Humana Press, New Jersey, 1995; andImmunoassay (ed. Diamandis and Christopoulos), Academic Press, New York,1996. In some aspects, the labeled cells are incubated with ahorseradish peroxidase (HRP)-conjugated anti-FLAG antibody at conditionsoptimal for antibody binding. HRP conjugated anti-FLAG antibodies arecommercially available from suppliers such as, but not limited to,Sigma-Aldrich (St. Louis, Mo.), Cell Signaling (Danvers, Mass.), ProteinMods (Madison, Wis.), Prospec Bio (East Brunswick, N.J.). Theantibody-labeled cells are exposed to a luminol substrate that isoxidized by HRP in a chemiluminescent reaction. The light-emittingreaction is detectable using imaging systems such as, but not limited toOdyssey Fc System (LI-COR). In other aspects, the FLAG taggedbiomolecules are extracted from the cells using methods known to thoseskilled in the art. Descriptions of methods for the isolation ofbiomolecules and the detection of FLAG-tagged biomolecules can be foundin references such as, but not limited to, Current Protocols inMolecular Biology (ed. Ausubel et al.), Wiley, New Jersey, 2011; andCurrent Protocols in Immunology (ed. Coligan et al.), Wiley, New Jersey,2011.

Example 44

Example 44 illustrates the compounds of the present invention withtechnology similar to Rutjes (cf. ChemBioChem 2007, 8, 1504-1508) usingcopper-free click chemistry reaction conditions as shown in Scheme 3.This example illustrates a method of covalently binding a near-infrareddye to selectively modified biomolecules that are expressed by cells.Non-limiting examples of applications of the methods described here inare tumor imaging, glycan labeling, in vivo labeling, cell surfacemodification, The method is based on a tandem [3+2]cycloaddition-retro-Diels-Alder ligation method that results in a stable1,2,3-triazole linkage.

In this example, the reaction is carried-out with IRDye 800CW as asample dye, but this proceeding is also applicable toe the dye(s) of thepresent invention.

Scheme 3 illustrates a method of linking a near-infrared dye to anazido-labeled biomolecule. In some embodiments, firstly, anoxanorbornadiene is coupled to a near-infrared dye (e.g., IRDye 800CW)via an amidation reaction to generate an IRDye800CW-oxanorbornadiene.Next, the IRDye800CW-oxanorbornadiene reagent is incubated with cellsexpressing an azido labeled biomolecule, thereby creating a “click”reaction. Typically, azido labeled cells are generated following methodsdescribed in Example 43. The tandem [3+2] cycloaddition andretro-Diels-Alder reactions generate a furan molecule and a triazolelinkage between the biomolecule and the dye, thereby labeling targetedcells with a dye.

In certain embodiments, a IRDye800CW-oxanorbornadiene reacts with aselectively modified azido-biomolecule on cells in an animal. TheIRDye800CW-oxanorbornadiene can be administered (e.g., injection, oral,transdermal and topical) to an animal. In some instances, theIRDye800CW-labeled cells and biomolecules are monitored in the animalusing an infrared detection system (e.g., Pearl Imager). In otherinstances, cells and tissues from the animal are harvested and analyzedusing techniques known to those in skilled in the art, such as, but notlimited to ELISA, FLISA, Western, histology, immunocytochemistry, andimaging. Methods including protocols are available in references suchas, but not limited to Current Protocols in Molecular Biology (ed.Ausubel et al.), Wiley, New Jersey, 2011; Current Protocols in ProteinScience (ed. Coligan et al.), Wiley, New Jersey, 2011; and CurrentProtocols in Immunology (ed. Coligan et al.), Wiley, New Jersey, 2011.In some instances, the labeled biomolecules and cells are monitoredusing techniques described in Example 43.

In certain embodiments, the labeled biomolecule is detected andidentified using methods such as, but not limited to high-performanceliquid chromatography (HPLC) and liquid chromatography-mass spectrometry(LC-MS). Methods of detecting dye-labeled cells and biomolecules aredescribed in references, for example, Peptide Characterization andApplication Protocols (ed. Fields), Humana Press, New Jersey, 2007;Sample Preparation in Biological Mass Spectrometry (ed. Ivanov andLazarev), Springer, New York, 2010; and Proteomic Biology Using LC-MS:Large Scale Analysis of Cellular Dynamics and Function, (ed. Takahashiand Isobe), Wiley, New Jersey, 2008. In some instances, the labeledbiomolecule is a constituent of a molecular complex and methods ofdissociating, separating or modifying the complex are used prior toperforming methods for detecting and identifying the individual labeledpeptides. Examples include, but are not limited to LC-MS with peptidemass fingerprinting and tandem MS (LC-MS/MS).

Example 45

Example 45 illustrates the synthesis of sodium3,3T-(2-((E)-2-((E)-3-((E)-2-(3-(6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl)-1,1-dimethyl-7-sulfonato-1H-benzo[e]indol-2(3H)-ylidene)ethylidene)-2-(2-fluorophenyl)cyclohex-1-enyl)vinyl)-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indolium-5-ylazanediyl)dipropane-1-sulfonate(34).

Compound 34 was prepared by combining 60 mg of compound 25a, 32 mg ofN,N′-disuccinimidyl carbonate, 10.8 μL of N,N-diisopropylethylamine, and3 mL of DMSO. The mixture was stirred at room temperature for 30minutes, precipitated into diethyl ether, and then purified byreverse-phase C18 chromatography using acetonitrile/water, yielding 29.8mg of blue-green solid.

Example 46

Example 46 illustrates the synthesis of a TFA salt of thefluorescence-quenching dye sodium1-(6-(6-aminohexylamino)-6-oxohexyl)-2-((E)-2-((E)-3-((E)-2-(3,3-dimethyl-5-sulfonato-1-(4-sulfonatobutyl)indolin-2-ylidene)ethylidene)-2-(4-sulfonatophenoxy)cyclohex-1-enyl)vinyl)-3,3-dimethyl-3H-indolium-5-sulfonate(IRDye 800CW-Hexamethylenediamine, 35a).

The IRDye 800CW NHS ester 36 (300 mg, 0.3 mmol) was reacted withN-Boc-1,6-hexanediamine (167.5 mg, 0.8 mmol) in 10 ml DMSO under N₂ for2 hours, then the mixture was precipitated by ether and purified by C18flash chromatography. The resulting material was then deprotected by TFAto afford the compound 35b as a green solid that was used withoutfurther purification.

Example 47

Example 47 illustrates the synthesis of sodium3,3′-((E)-2-((E)-2-(3-((E)-2-(3-(6-(6-(6-(2-((E)-2-((E)-3-((E)-2-(3,3-dimethyl-5-sulfonato-1-(4-sulfonatobutyl)indolin-2-ylidene)ethylidene)-2-(4-sulfonatophenoxy)cyclohex-1-enyl)vinyl)-3,3-dimethyl-5-sulfonato-3H-indolium-1-yl)hexanamido)hexylamino)-6-oxohexyl)-1,1-dimethyl-7-sulfonato-1H-benzo[e]indolium-2-yl)vinyl)-2-(2-fluorophenyl)cyclohex-2-enylidene)ethylidene)-3,3-dimethyl-1-(3-sulfonatopropyl)indolin-5-ylazanediyl)dipropane-1-sulfonate(800CW-NH—(CH₂)₆—NH-Compound 25a, 8-atom linker; 37).

A solution of IRDye 800CW-Hexamethylenediamine.TFA (35b) (64 μg,5.0×10⁻² μmol) and N,N-diisopropylethylamine (0.1 4, 5.7×10⁻¹ μmol) inanhydrous dimethyl sulfoxide (200 μL) was added to a vessel containingcompound 34 (100 μg, 7.7×10⁻² μmol). The reaction mixture was agitatedfor 30 seconds and then allowed to proceed at ambient temperature for 2hours. After HPLC analysis showed the complete consumption of800CW-Hexamethylenediamine.TFA, the reaction mixture was precipitatedinto dry diethyl ether. The ethereal layer was decanted and the crudeproduct residue was purified by reverse-phase HPLC to afford the product37 as a teal solid. The exact yield was not determined. UV/Vis(acetonitrile/water=1:1) λ_(max)1=778 nm, λ_(max)2=830 nm; LRMS(ES/water), m/z calculated for C₁₀₇H₁₃₃FN₇O₂₇S₈ [M+H]⁺2222.74. found742.5 [M+3H]³⁺, 1113.1 [M+H]²⁺.

Example 48

Example 48 illustrates the synthesis of sodium3,3′-((E)-2-((E)-2-((3-((E)-2-(3-(27-(2-((E)-2-((E)-3-((E)-2-(3,3-dimethyl-5-sulfonato-1-(4-sulfonatobutyl)indolin-2-ylidene)ethylidene)-2-(4-sulfonatophenoxy)cyclohex-1-enyl)vinyl)-3,3-dimethyl-5-sulfonato-3H-indolium-1-yl)-6,22-dioxo-11,14,17-trioxa-7,21-diazaheptacosyl)-1,1-dimethyl-7-sulfonato-1H-benzo[e]indolium-2-yl)vinyl)-2-(2-fluorophenyl)cyclohex-2-enylidene)ethylidene)-3,3-dimethyl-1-(3-sulfonatopropyl)indolin-5-ylazanediyl)dipropane-1-sulfonate(800CW-NH-(PEG)₂-NH-Compound25a, 15-atom linker; 38).

This compound was prepared in a manner similar to 37 (Example 47) fromthe corresponding IRDye 800CW-NH-(PEG)₂-NH₂.TFA (32). The exact yieldwas not determined. UV/Vis (acetonitrile/water=1:1) λ_(max)1=778 nm,λ_(max)2=830 nm; (ES/water), m/z calculated forC₁₁₁H₁₄₁FN₇O₃₀S₈[M+H]⁺2326.75. found 776.8 [M+3H]³⁺ and 1164.1 [M+2H]²⁺.

Example 49

Example 49 illustrates the synthesis of sodium3,3′4(E)-2-((E)-2-(3-((E)-2-(3-(49-(2-((E)-2-((E)-3-((E)-2-(3,3-dimethyl-5-sulfonato-1-(4-sulfonatobutyl)indolin-2-ylidene)ethylidene)-2-(4-sulfonatophenoxy)cyclohex-1-enyl)vinyl)-3,3-dimethyl-5-sulfonato-3H-indolium-1-yl)-6,44-dioxo-10,13,16,19,22,25,28,31,34,37,40-undecaoxa-7,43-diazanonatetracontyl)-1,1-dimethyl-7-sulfonato-1H-benzo[e]indolium-2-yl)vinyl)-2-(2-fluorophenyl)cyclohex-2-enylidene)ethylidene)-3,3-dimethyl-1-(3-sulfonatopropyl)indolin-5-ylazanediyl)dipropane-1-sulfonate(800CW-NH-dPEG₁₁-NH-Compound25a, 37-atom linker; 39).

This compound was prepared in a manner similar to 52 (Example 62) fromthe corresponding IRDye 800CW-NH-dPEG₁₁®—NH₂.TFA, which was prepared bya method analogous to that used to prepare compound 32 fromBoc-NH-dPEG₁₁®-NH₂ (Quanta Biodesign, Inc.) (Example 39). The exactyield was not determined. UV/Vis (acetonitrile/water=1:1) λ_(max)1=777nm, λ_(max)2=830 nm; LRMS (ES/water), m/z calculated forC₁₂₅H₁₆₉FN₇O₃₈S₈ [M+H]′ 2650.93. found 884.9 [M+3H]³⁺ and 1337.6[M+2H]²⁺.

Example 50

Example 50 illustrates the synthesis of a strainedcycloalkyne-containing a compound 25a derivative for click chemistry(Compound 25a-DBCO, 40).

This compound was prepared in a manner similar to that used for 37(Example 47) from the commercially available DBCO-Amine. To a solutionof compound 25a (Example 25, 0.8 mg, 6.1×10⁻⁴ mmol) andN,N-diisopropylethylamine (0.001 mL, 5.7×10⁻³ mmol) in anhydrousdimethylsulfoxide (0.8 mL) was added DBCO-Amine (from Jena Bioscience,0.5 mg, 1.8×10⁻³ mmol) in one portion. The reaction was allowed toproceed at ambient temperature for 2 h, with periodic agitation at15-min intervals. After HPLC analysis indicated complete consumption of25a, the crude product was precipitated in anhydrous diethyl ether (10mL). The ethereal supernatant was decanted and the precipitate waspurified by prep-HPLC to afford the desired product 40 as a teal solid(0.8 mg, 90%). UV/Vis (acetonitrile/water=1:1) λ_(max)=790 nm; LRMS(ES/water), m/z calculated for C₇₃H₈₁FN₅O₁₄S₄ [M+H]⁺1398.46. found1398.5.

Example 51

Example 66 illustrates the synthesis of an azide-containing compound 25aderivative for click chemistry (Compound 25a-PEG-Azide, 41).

This compound was prepared in a manner similar to that used for 37(Example 47) from commercially available11-azido-3,6,9-trioxaundecan-1-amine. The exact yield was notdetermined. UV/Vis (acetonitrile/water=1:1) λ_(max)=790 nm; LRMS(ES/water), m/z calculated for C₆₃H₈₃FN₇O₁₆S₄ [M+H]′ 1340.48. found1340.5 and 671.0 [M+2H]²⁺.

Example 52

Example 67 illustrates the click chemistry synthesis of a compound 25adimer (42).

A solution of cycloalkyne 40 (100 μg, 6.8×10⁻² mop in water (100 μL) wasmixed with a solution of azide 41 (6.8×10⁻² μg, 7.1×10⁻² mol) in water(100 μL). The reaction mixture was agitated for 30 seconds and thenallowed to proceed at ambient temperature for 2 hours. After HPLCanalysis showed the complete consumption of 41, the reaction mixture wasdirectly purified by reverse-phase HPLC to afford 42 as a blue solid.The exact yield was not determined. UV/Vis (acetonitrile/water=1:1)λ_(max)=789 nm; LRMS (ES/water), m/z calculated for C₁₃₆H₁₆₃β2N12O₃₀S8[M+H]⁺ 2737.93. found 1368.7 [M+2H]²⁺.

Example 53

Example 53 illustrates the click chemistry synthesis of an IRDye800CW/compound 25a conjugate (800CW/Compound 25a Click Product 1, 58).

A solution of 40 (150 μg, 1.0×10⁻¹ mmol) in water (150 μL) was mixedwith a solution of 800CW-PEG-Azide (29, Example 36) (63 μg, 5.0×10⁻²mmol) in water (50 μL). The reaction mixture was agitated for 30 secondsand then allowed to proceed at ambient temperature for 2 hours. AfterHPLC analysis showed the complete consumption of 40, the reactionmixture was directly purified by reverse-phase HPLC to afford theproduct 43 as a teal solid that was a mixture of the twotriazole-cycloaddition regioisomers. The exact yield was not determinedUV/Vis (acetonitrile/water=1:1) λ_(max)1=778 nm, λ_(max)2=830; LRMS(ES/water), m/z calculated for C₁₂₇H₁₅₁FN₁₁O₃₁S₈ [M+H]⁺2600.83. found1299.6 [M−2H]²⁻.

Example 54

Example 54 illustrates the click chemistry synthesis of a second800CW/QC-2 conjugate (800CW/Compound 25a Click Product 2, 44).

A solution of 41 (150 μg, 1.0×10⁻¹ μmol) in water (150 μL) was mixedwith a solution of 800CW-DBCO (30. Example 37) (66 μg, 5.0×10⁻² μmol) inwater (50 μL). The reaction mixture was agitated for 30 seconds and thenallowed to proceed at ambient temperature for 2 hours. After HPLCanalysis showed the complete consumption of starting material, thereaction mixture was directly purified by reverse-phase HPLC to affordthe product 44 as a mixture of the two triazole cycloadditionregioisomers. UV/Vis (acetonitrile/water=1:1) λ_(max)1=778 nm,λ_(max)2=830 nm; LRMS (ES/water), m/z calculated for C₁₂₇H₁₅₁FN₁₁O₃₁S₈[M+H]⁺2600.83. found #1300.1 [M−2H]²⁻.

Example 55

Example 55 illustrates a trityl-protected version of a sampleheterocyclic dye (compound 10 from U.S. Provisional Patent Appl. No.61/405,158) HETD (HETD-NH-(PEG)₂-NH-Trt, 45).

A solution ofO-(N-trityl-3-aminopropyl)-O′-(3-aminopropyl)-diethyleneglycol(Trt-NH-PEG₂-NH₂, 1.0 mg, 2.2×10⁻³ mmol) and N,N-diisopropylethylamine(0.001 mL, 5.7×10⁻³ mmol) in anhydrous dimethyl sulfoxide (0.2 mL) wasadded to a reaction vessel containing HETD NHS ester (1.0 mg, 1.0×10⁻³mmol; compound 11 from U.S. Provisional Patent Appl. No. 61/405,158).The reaction was allowed to proceed at ambient temperature for 2 hours,with periodic vortexing at 15-minute intervals. After HPLC analysisshowed complete consumption of HETD NHS ester, the reaction wasprecipitated into anhydrous diethyl ether. The ethereal layer wasdecanted and the crude product was purified by HPLC. Fractionscontaining the presumed HETD-NH-PEG₂-NH-Trt in ≧95% purity were combinedand concentrated in vacuo to afford a blue film; the yield was presumedto be quantitative.

Example 56

Example 56 illustrates another derivative of HETD(HETD-NH-(PEG)₂-NH₂.2TFA, 46).

To a flask containing HETD-NH-PEG₂-NH-Trt (1.3 mg, 8.6×10⁻⁴) was added asolution of trifluoroacetic acid in dichloromethane (TFA/CH₂Cl₂=1:3, 5.0mL). The purple reaction was briefly swirled and allowed to proceed atambient temperature for 30 minutes. The volatiles were removed in vacuoand the residuals were treated again with TFA/CH₂Cl₂ (1:3, 5.0 mL) for30 minutes. After removing the volatiles in vacuo, the residuals werewashed with anhydrous diethyl ether. The ethereal layer was decanted,and the product 46 was used without further purification; the yield waspresumed to be quantitative. UV/Vis (methanol) λ_(max)=676 nm; LRMS(water) m/z calculated for 1064.4 [M+H]⁺. found 1064.6, 532.9 [M+2H]²⁺.

Example 57

Example 57 illustrates the synthesis of a phosphine HETD derivative(HETD-PEG-Phosphine, 47a).

To a solution of HETD-NH-(PEG)₂-NH₂.2TFA (1.3 mg, 1.0×10⁻³ mmol) inanhydrous dimethyl sulfoxide (0.2 mL) was added NHS-Phosphine (1.0 mg,2.2×10⁻³ mmol, ThermoScientific/Pierce) followed byN,N-diisopropylethylamine (0.001 mL, 5.7×10⁻³ mmol). The reaction wasallowed to proceed at ambient temperature for 2 hours, with periodicagitation at 15-minute intervals. After HPLC analysis showednear-complete consumption of the HETD-NH-(PEG)₂-NH₂.2TFA, the reactionwas precipitated into anhydrous diethyl ether. The ethereal layer wasdecanted and the crude product was purified by HPLC. Fractionscontaining the presumed HETD-PEG-Phosphine in ≧95% purity were combinedand concentrated in vacuo to afford a blue solid (0.7 mg, 51% based onHETD-NH-(PEG)₂-NH₂.2TFA); UV-Vis (methanol) λ_(max)=676 nm; LRMS (water)m/z calculated for 1410.4 [M+H]⁺. found 705.8 [M+2H]²⁺.

Example 58

Example 58 illustrates a phosphine oxide HETD derivative(HETD-PEG-Phosphine Oxide, 47b).

This compound was isolated as a substantial byproduct from the synthesisof HETD-PEG-Phosphine (47a). This byproduct is nonfunctional and causesbackground problems. The compound is a blue solid (0.2 mg, 18% basedHETD-NH-(PEG)₂-NH₂.2TFA); UV-Vis (methanol) λ_(max)=676 nm; LRMS (water)m/z calculated for 1426.4 [M+H]⁺. found 1426.5, 713.9 [M+2H]²⁺.

Example 59

Example 59 illustrates the click chemistry synthesis of anIRDye®HETD/Compound 25a conjugate (HETD-Compound 25a Click Product, 48).

To a solution of azide 41 (150 μg, 1.1×10⁻¹ mmol) in water (150 μL) wasadded a solution of HETD-PEG-Phosphine (47a) (92 μg, 6.3×10⁻² μmol) inwater (50 μL). The reaction was allowed to proceed at ambienttemperature for 1 hour, then maintained at 40° C. for 3 hours. AfterHPLC analysis showed complete consumption of HETD-PEG-Phosphine, thereaction mixture was filtered and directly purified by HPLC. Fractionscontaining the presumed HETD-Compound 25a Click Product 48 were combinedand concentrated in vacuo. The exact yield was not determined. UV/Vis(acetonitrile/water=1:1) λ_(max)1=778 nm, λ_(max)2=677 nm; LRMS(ES/water), m/z calculated for C₁₂₃H₁₅₀ClN₉O₃₃PS₇ [M+H]⁺2570.8. found858.5 [M+3H]³⁺.

Example 60

Example 6 illustrates the synthesis of the fluorescence-quenching dyesodium(E)-2-((E)-2-(3-((E)-2-(5-(bis(3-sulfonatopropyl)amino)-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indolium-2-yl)vinyl)-5-carboxy-2-(2-fluorophenyl)cyclohex-2-enylidene)ethylidene)-1,1-dimethyl-3-(3-sulfonatopropyl)-2,3-dihydro-1H-benzo[e]indole-6,8-disulfonate(49).

Compound 49 was prepared by combining 200 mg of compound 50, 31 mg of2-fluorophenylboronic acid, 12.7 mg of Pd(PPh₃)₄, 36.1 mg of sodiumacetate, 800 μL of 2-methoxyethanol, and 8 mL of water. The mixture washeated at reflux for 45 minutes under a nitrogen atmosphere. To thereaction was then added 1 mL of 10% sulfuric acid solution and refluxwas continued for 90 minutes. The compound was purified by reverse-phaseC18 chromatography using acetonitrile/water, yielding 13 0 mg ofblue-green product. Absorbance: λ_(Water)=779 nm.

Example 61

Example 61 illustrates the synthesis of an NHS ester of compound 49(51).

Compound 51 was prepared by combining 130 mg of compound 49, 72 mg ofN,N′-disuccinimidyl carbonate, 25 μL of N,N-diisopropylethylamine, and 8mL of DMSO. The mixture was sonicated at room temperature for 120minutes, precipitated into diethyl ether, and then purified byreverse-phase C18 chromatography using acetonitrile/water, yielding 80mg of blue-green product.

Example 62

Example 62 illustrates the synthesis of an IRDye 800CW/Compound 64conjugate (IRDye 800CW-NH-(PEG)₂-NH-Compound 64, 15-atom linker; 52).

A solution of IRDye 800CW-(PEG)₂-NH₂.TFA salt (32, Example 39) (69 μg,5.0×10⁻² μmol) and N,N-diisopropylethylamine (0.1 μL, 5.7×10⁻¹ μmol) inanhydrous dimethyl sulfoxide (200 μL) was added to a vessel containingcompound 51 (100 μg, 7.3×10⁻² μmol). The reaction mixture was agitatedfor 30 seconds and then allowed to proceed at ambient temperature for 2hours. After HPLC analysis showed the complete consumption of the IRDye800CW-(PEG)₂-NH₂.TFA salt, the reaction mixture was precipitated intodry diethyl ether. The ethereal layer was decanted and the crude productresidue was purified by reverse-phase HPLC to afford the product 52 as ateal solid. The exact yield was not determined. UV/Vis(acetonitrile/water=1:1) λ_(max)1=778 nm, λ_(max)2=830 nm; LRMS(ES/water), m/z calculated for C₁₀₉H₁₃₇FN₇O₃₆S₁₀ [M+H]⁺2458.62. found820.8 [M+3H]³⁺.

Example 63

Example 63 illustrates the synthesis of another IRDye 800CW/compound 49conjugate (IRDye 800CW-NH-dPEG₁₁-NH-Compound 49, 37-atom linker; 53).

This compound was prepared in a manner similar to 39 (Example 49) fromthe corresponding IRDye 800CW-NH-dPEG₁₁®—NH₂.TFA, which was prepared bya method analogous to 32 (Example 39) from Boc-NH-dPEG₁₁®—NH₂ (QuantaBiodesign, Inc.). The exact yield was not determined. UV/Vis(acetonitrile/water=1:1) λ_(max)1=777 nm, λ_(max)2=829 nm; LRMS(ES/water), m/z calculated for C₁₂₃H₁₆₅FN₇O₄₄S₁₀ [M+H]⁺2782.80. found928.5 [M+3H]³⁺.

Example 64

Example 64 illustrates the synthesis of a strainedcycloalkyne-containing compound 49 derivative for click chemistry(Compound 49-DBCO; 54).

This compound was prepared in a manner similar to that used for 40(Example 50) from the commercially available DBCO-Amine. The exact yieldwas not determined. UV/Vis (acetonitrile/water=1:1) λ_(max)=799 nm; LRMS(ES/water), m/z calculated for C₇₁H₇₇FN₅O₂₀S₆ [M+H]⁺1530.34. found 765.9[M+2H]²⁺.

Example 65

Example 65 illustrates the synthesis of an azide-containing a compound49 derivative for click chemistry (Compound 49-PEG-Azide, 55).

This compound was prepared in a manner similar to that used for 41(Example 51) from commercially available11-azido-3,6,9-trioxaundecan-1-amine. The exact yield was not determinedUV/Vis (acetonitrile/water=1:1) λ_(max)=799 nm.; LRMS (ES/water), m/zcalculated for C₆₁H₇₉FN₇O₂₂S₆ [M+H]⁺1472.35. found 736.9 [M+2H]²⁺.

Example 66

Example 66 illustrates the synthesis of an IRDye 800CW/compound 49conjugate (IRDye 800CW/Compound 49 Click Product 1; 56).

This compound was prepared in a manner similar to that used for 43(Example 53) from cycloalkyne 54 and IRDye 800CW-PEG-Azide 29 (Example36). The exact yield was not determined, and the compound was a mixtureof the two cycloaddition regioisomers. UV/Vis (acetonitrile/water=1:1)λ_(max)1=778 nm, λ_(n), λ_(max)2=830 nm; LRMS (ES/water), m/z calculatedfor C₁₂₅H₁₄₇FN₁₁O₃₇S₁₀ [M+H]⁺2732.71. found 912.2 [M+3H]³⁺, 1365.6[M−2H]²⁻, 910.4 [M−3H]³⁻.

Example 67

Example 82 illustrates the synthesis of an IRDye 800CW/compound 49conjugate (IRDye 800CW/Compound 64 Click Product 2; 57).

This compound was prepared in a manner similar to that used for 43(Example 53) from Compound 64-PEG-Azide (55, Example 65) and IRDye800CW-DBCO (29, Example 36). The exact yield was not determined, and thecompound was a mixture of the two cycloaddition regioisomers. UV/Vis(acetonitrile/water=1:1) λ_(max)1=778 nm, λ_(max) 2=830 nm; LRMS(ES/water), m/z calculated for C ₁₂₅H₄₇F₁₁O₃₇S10 [M+H]⁺2732.71. found1367.6 [M+2H]²⁺, 912.2 [M+3H]³⁺, 1365.6 [M−2H]²⁻, 910.4 [M−3H]³⁻.

Example 68

Example 68 illustrates the synthesis of an strainedcycloalkyne-containing HETD derivative for click chemistry (HETD-DBCO,58).

This compound was prepared in a manner similar to that used for compound40 (Example 50) from the commercially available DBCO-Amine. The exactyield was not determined. UV/Vis (acetonitrile/water=1:1) λ_(max)=676nm; UV/Vis (acetonitrile/water=1:1) λ_(max)=676 nm; LRMS (ES/water), m/zcalculated for C₅₇H₅₉ClN₅O₁₁S₃ [M+H]⁺1120.30. found 1120.6. 560.9[M+2H]²⁺.

Example 69

Example 69 illustrates the synthesis of an azide-containing HETDderivative for click chemistry (HETD-PEG-Azide, 59).

This compound was prepared in a manner similar to that used for 41(Example 55) from the commercially available11-azido-3,6,9-trioxaundecan-1-amine. The exact yield was notdetermined. UV/Vis (acetonitrile/water=1:1) λ_(max)=676 nm; LRMS(ES/water), m/z calculated for C₄₇H₆₁ClN₇O₁₃S₃ [M+H]⁺1062.31. found1060.6 [M−H]⁻, 1082.6 [M+Na−2H]²⁻.

Example 70

Example 70 illustrates the synthesis of a HETD/compound 49 conjugate(HETD/Compound 49 Click Product 1; 60).

This compound was prepared in a manner similar to that used for compound43 (Example 53) from Compound 49-DBCO (54) (Example 64) andHETD-PEG-Azide (59)

(Example 69). The exact yield was not determined, and the compound was amixture of the two cycloaddition regioisomers. UV/Vis(acetonitrile/water=1:1) λ_(max)1=679 nm, λ_(max)2=799 nm; LRMS(ES/water), m/z calculated for C₁₁₈H₁₃₇ClFN₁₂O₃₃S₉ [M+H]⁺2591.65. found1295.0 [M−2H]²⁻, 863.1 [M−3H]³⁻.

Example 71

Example 71 illustrates the synthesis of another HETD/compound 49conjugate (HETD/Compound 49 Click Product 2; 61).

This compound was prepared in a manner similar to that used for 43(Example 53) from Compound 49-PEG-Azide (55, Example 65) and HETD-DBCO(58, Example 68). The exact yield was not determined, and the compoundwas a mixture of the two cycloaddition regioisomers. UV/Vis(acetonitrile/water=1:1) λ_(max)1=679 nm, λ_(max)2=799 nm; LRMS(ES/water), m/z calculated for C₁₁₈H₁₃₇ClFN₁₂O₃₃S₉ [M+H]⁺2591.65. found1295.6 [M−2H]²⁻.

Example 72

Objective:

For some dye scaffolds, the final Suzuki coupling step to install a2,4,6-trifluorophenyl polyene substituent has a low chemical yield(˜10%). In this experiment, the yields and properties of additionalpolyene substituents were evaluated.

Design Principle:

We analyzed the properties of a dye scaffold and related compounds andthe corresponding substitution effects on the excitation and emissionwavelengths. The small size of fluorine is unlikely to produce stericeffects, therefore, we explored the electronic effects of fluorine andrelated substitutions. The Hammett equation (σ-value) is an importanttool to understand the electronic effects of the substituents,therefore, we explored the correlation of the wavelength (λ_(MeOH)) andthe σ-value (vide infra):

TABLE 5 Absorption (λ) and σ- Value for Related Dyes λ_(MeOH) λ_(MeOH) X(nM) σ X (nM) σ

  62a 766

  62f 782 σ_(o(F)) = 0.24 σ_(o(F)) = 0.24 σ_(p(COOH)) = . . .

  62b 772 σ_(o(F)) = 0.24

  62g 782 σ_(o(Cl)) = 0.2 σ_(o(Cl)) = 0.2

  62c 782 σ_(o(F)) = 0.24 σ_(o(F)) = 0.24

  62h 772 σ_(o(F)) = 0.24 σ_(o(F)) = 0.24 σ_(p(OMe)) = −0.27

  62d 785 σ_(o(F)) = 0.24 σ_(o(F)) = 0.24 σ_(p(F)) = 0.06

  62i 779 σ_(o(F)) = 0.24 σ_(o(F)) = 0.24 σ_(m(-O-nBu)) = 0.10

  62e 789 σ_(o(F)) = 0.24 σ_(o(F)) = 0.24 σ_(p(F)) = 0.06 σ_(m(-O-Bu)) =0.10

  62j 790 σ_(o(F)) = 0.24 σ_(m(F)) = 0.34 σ_(m(-O-nBu)) = −0.24

The above mentioned examples indicate that the electronic effects play amajor role in the corresponding dye's optical properties and acorrelation between the wavelength (λ_(MeOH))and the σ-value can bemade. Therefore, a low yielding Suzuki coupling step can be circumventedby replacing 2,4,6-trifluoro-substitution with 2,3-difluoro substitutiononly, as both substitution patterns should lead to similar electroniceffects. The detailed synthetic protocol and optical properties arediscussed below.

TABLE 6 Summary of Photophysical Properties     Abs Max PBS/      Em_(Max) $\mspace{31mu} {\begin{matrix}\frac{{Fl}\mspace{14mu} {Intensity}\mspace{14mu} {of}\mspace{14mu} X}{{Fl}\mspace{14mu} {Intensity}\mspace{14mu} {of}\mspace{14mu} 800\mspace{14mu} {CW}} \\\left( {0.1\mspace{14mu} \mu \; M\mspace{14mu} {solutions}\mspace{14mu} {in}\mspace{14mu} 1{XPBS}} \right)\end{matrix}\quad}$ Quantum Yield_(Stnd) = IRDye 800 CW X = Methanol PBSFluorimeter Odyssey (QY = 0.07)

  62k 773 nm/ 781 nm 790 nm 100% 104% 0.060 N/A (IRDye 775 790 100% 100%0.07  IRDye 800 CW) nm (in nm PBS only)

Synthesis of 2,3-Difluorophenyl Dye (62k)

To a 100-mL pressure tube with a magnetic stir-bar were added thechloro-precursor dye (62m) (208 mg, 0.218 mmol), 2,3-difluorophenylboronic acid (84.8 mg, 0.537 mmol),tetrakis(triphenylphosphine)palladium(0) (23.2 mg, 0.0221 mmol), sodiumacetate (75.3 mg, 0.918 mmol), ultra-pure water (6 mL) and2-methoxyethanol (1 mL). The pressure tube was purged with argon for 2min and was heated at 115° C. for 1 h. The reaction mixture was cooleddown to ambient temp. and the solvent was removed under reducedpressure. The reaction mixture was triturated with methylene chloride(50 mL), and the product 62k was purified on C18 reverse-phase silicausing water:acetonitrile as the eluent (66.5%, 99.3% HPLC purity at 780nm); mass. spec. obs. 965.6, expected 965.2.

In an alternative version of the procedure, to a 100-mL pressure tubewith a magnetic stir-bar were added the chloro dye (62m) (207 mg, 0.223mmol), 2,3-difluorophenyl boronic acid (107 mg, 0.675 mmol),tetrakis(triphenylphosphine)-palladium(0) (24.9 mg, 0.0215 mmol), sodiumacetate (55.4 mg, 0.675 mmol), ultra-pure water (6 mL) and2-methoxyethanol (1 mL). The pressure tube was purged with argon for 2min and was heated at 115° C. for 45 min. The reaction mixture wascooled down to ambient temp. and the solvent was removed under reducedpressure. The reaction mixture was triturated with methylene chloride(50 mL), and the product 62k was purified on C18 reverse-phase silicausing water:acetonitrile as eluent.

Example 73

Compound 63a was prepared by adding compound 63b (434.0 mg, 0.4060 mmol;which can be prepared from compounds 2 and 6 by the method of Example 7,follow by ester hydrolysis by a conventional method) to an oven-driedpressure tube with a magnetic stir-bar with 2,3-difluorophenylboronicacid (102.0 mg, 0.6460 mmol), sodium acetate (80.0 mg, 0.646 mmol),tetrakis triphenyl phosphine palladium(0) (45.0 mg, 39.0 μmol), water(12 mL) and methoxymethanol (2 mL). The pressure tube was purged withnitrogen before heating the mixture at 115° C. for 2 h. The reactionmixture was neutralized with 1(N) hydrochloric acid (2404). After HPLCanalysis showed complete consumption of the presumed mixed carbonateintermediate, the reaction mixture was concentrated in vacuo to afford acrude residue. The reaction mixture was triturated the crude dye with 25mL of dichloromethane. The dichloromethane layer was removed and theresidue was purified by reverse-phase flash chromatography to furnishthe desired product63 as a green solid (306.3 mg, 75%). UV/Vis(methanol) λmax=770 nm; LRMS (ES/acetonitrile), m/z calculated forC₄₅H₅₀F₂N₂O₁₄S₄ [M+H]⁺1009.3. found 1009.2.

Example 74

Compound 64 was prepared by adding compound 62m (387.1 mg, 0.4060 mmol)to an oven-dried pressure tube with a magnetic stir-bar with, 1-carboxyl2,3-difluoro boronic acid (130.4 mg, 0.6460 mmol), sodium acetate (80.0mg, 0.646 mmol), tetrakis triphenyl phosphine palladium(0) (45.0 mg, 39μmol), water (12 mL) and methoxymethanol (2 mL). The pressure tube waspurged with nitrogen before heating the mixture at 115° C. for 2 h. Thereaction mixture was neutralized with 1 N hydrochloric acid (240 μL).After HPLC analysis showed complete consumption of the presumed mixedcarbonate intermediate, the reaction mixture was concentrated in vacuoto afford a crude residue. The reaction mixture was triturated the crudedye with 25 mL of dichloromethane. The dichloromethane layer was removedand the residue was purified by reverse-phase flash chromatography tofurnish the desired product 6 as a green solid (294 mg, 72%). UV/Vis(methanol) λ_(max=)772 nm; LRMS (ES/acetonitrile), m/z calculated forC45H₅₀F₂N₂O₁₄S4 [M+H]⁺ 1009.6. found 1009.4.

Example 75

Compound 65 was prepared by adding compound 62 m (387.1 mg, 0.4060 mmol)to an oven-dried pressure tube with a magnetic stir-bar with boronicacid (148.6 mg, 0.6460 mmol), sodium acetate (80.0 mg, 0.646 mmol),tetrakis triphenyl phosphine palladium(0) (45.0 mg, 39 mmol), water (12mL) and methoxymethanol (2 mL). The pressure tube was purged withnitrogen before heating the mixture at 115° C. for 2 h. The reactionmixture was neutralized with 1 N hydrochloric acid (240 μL). After HPLCanalysis showed complete consumption of the presumed mixed carbonateintermediate, the reaction mixture was concentrated in vacuo to afford acrude residue. The reaction mixture was triturated the crude dye with 25mL of dichloromethane. The dichloromethane layer was removed and theresidue was purified by reverse-phase flash chromatography to furnishthe desired product 6 as a green solid (319.9 mg, 76%). UV/Vis(methanol) λ_(max=)776 nm; LRMS (ES/acetonitrile), m/z calculated forC₄₇H₅₄F₂N₂O₁₄S4 [M+H]⁺ 1038.4. found 1038.4.

Example 76

Compound 66 was prepared by adding compound 64 (430 mg, 0.40 mmol) to anoven-dried round-bottomed flask containing magnetic stir-bar and septumunder nitrogen, followed by anhydrous DMSO (10 mL) anddiisopropylethylamine (206 mg, 2.40 mmol). The flask was placed in asonicator for 5 min followed by addition of N,N′-disuccinimidylcarbonate (612 mg, 2.40 mmol). The reaction mixture was stirred for 2 hat ambient temperature. After HPLC analysis showed complete consumptionof the starting material, a solution of methyl 6-amino hexanoate (63.8mg, 0.44 mmol) in anhydrous DMSO (1 mL) was added, and the reactionmixture was stirred for an additional 2 h. The reaction mixture wasconcentrated in vacuo to afford a crude residue. The residue wasdissolved in water (5 mL) and a 1 N hydrochloric acid (250 μL) was addeddropwise. The reaction mixture was stirred at ambient temperature for 2h. The solution was concentrated under reduced pressure and theresulting green residue was purified by reverse-phase flashchromatography to furnish the desired product as a green solid (278.5mg, 62%). UV/Vis (methanol) λ_(max)=773 nm; LRMS (ES/acetonitrile), m/zcalculated for C₅₁H₆₁F₂N₃O₁₅S4 [M+H]⁺ 1123.1. found 1123.3.

Example 77

5-Carboxymethyl-2,3,3-trimethylindoline (67) is prepared either by themethods of Southwick et al., Org. Prep. Proceed. Int., 20, 274-84,(1989) or alternatively by the method of U.S. Pat. No. 6,133,445.

Example 78

Compound 68 is prepared analogously to compound 1 except with5-carboxymethyl-2,3,3-trimethylindoline (compound 67) as the startingmaterial.

Example 79

Compound 69 is prepared analogously to compound 7 except that compound68, compound 5, and compound 1 are starting materials.

Example 80

Compound 70 is prepared analogously to compound 11 except that compound69 and 2,4,6-trifluorophenylboronic acid are used as starting materials.

Example 81 Comparison of Dye Emission Maxima and Quenching

Solutions of dye-linker standards and dye-linker-quencher samples werediluted into PBS buffer (pH 7.4) to give a dye-specific absorbance lessthan 0.2 AU. The fluorescence spectra of each dilution were then takenat a consistent excitation wavelength (670 nm for HETD and 770 nm forIRDye 800CW). The emission spectra were collected from 680-1000 nm forthe HETD samples and 780-1000 nm for the IRDye 800CW samples.

Quencher Example Emission Percent Version Sample Name Number MaximumQuenching Compound HETD-PHOS-OX Example 3020520 25a (ref) (47b) 58HETD-Compound Example 1054060 65.1 25a -Click 1 (48) 59 IRDye800CW-PEG2- Example 1343900 NH2 (ref) (32) 39 IRDye 800CW-PEG2- Example385983 71.3 Compound 25a (38) 48 IRDye 800CW-C6- Example 519267 61.4Compound 25a (37) 47 IRDye 800CW- N/A 1272820 PEG11-NH2 (ref) IRDye800CW- Example 438981 65.5 PEG11-Compound 49 25a (39) IRDye 800CW-PEG-Example 1245060 N3 (ref) (29) 36 IRDye 800CW- Example 550761 55.8Compound 25a -Click 53 1 (43) IRDye 800CW- Example 1203780 DBCO (ref)(30) 37 IRDye 800CW- Example 424840 64.7 Compound 25a -Click 54 2 (44)Compound HETD-PHOS-OX Example 3020520 49 (ref) (47b) 58 HETD-Compound49- Example 278958 90.8 Click 1 (75) 85 HETD-Compound 49- Example 49922583.5 Click 2 (76) 86 IRDye 800CW-PEG2- Example 1343900 NH2 (ref) (32) 39IRDye 800CW-PEG2- Example 58878 95.6 Compound 49 (52) 62 IRDye 800CW-N/A 1272820 PEG11-NH2 (ref) IRDye 800CW- Example 128324 89.9PEG11-Compound 49 63 (53) IRDye 800CW-PEG- Example 1245060 N3 (ref) (29)36 IRDye 800CW- Example 43472 96.5 Compound 49-Click 1 66 (56) IRDye800CW- Example 1203780 DBCO (ref) (30) 37 IRDye 800CW- Example 6793094.4 Compound 49-Click 2 67 (57)

All publications, patents and patent applications and cited in thisspecification including Attorney Docket Number 020031-011910PC, filed oneven date herewith are herein incorporated by reference as if eachindividual publication, patent or patent application were specificallyand individually indicated to be incorporated by reference. Although theforegoing invention has been described in some detail by way ofillustration and example for purposes of clarity of understanding, itwill be readily apparent to those of ordinary skill in the art that, inlight of the teachings of this application, that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Additional background regarding click chemistry is included in thereferences that are shown below:

-   1. Ghosh, A. K.; Duong, T. T.; McKee, S. P.; Thompson, W. J.    “N,N′-disuccinimidyl carbonate: a useful reagent for    alkoxycarbonylation of amines.” Tetrahedron Lett. 1992, 33,    2781-2784.-   2. Ghosh, A. K.; McKee, S. P.; Duong, T. T.; Thompson, W. J. “An    efficient synthesis of functionalized urethanes from azides.” Chem.    Commun. 1992, 1308-13010.-   3. Højfeldt, J. W.; Blakskjær, P.; Gothelf, K. V. “A Cleavable    Amino-Thiol Linker for Reversible Linking of Amines to DNA.” J. Org.    Chem. 2006, 71, 9556-9559.-   4. Bertozzi, C. R.; Bednarski, M. D. “The synthesis of    heterobifunctional linkers for the conjugation of ligands to    molecular probes.”J. Org. Chem. 1991, 56, 4326-4329.-   5. Schwabacher, A. W.; Lane, J. W.; Scheisher, M. W.; Leigh, K. M.;    Johnson, C. W. “Desymmetrization Reactions: Efficient Preparation of    Unsymmetrically Substituted Linker Molecules.” J. Org. Chem. 1998,    63, 1727-1729.-   6. Website: http://www.baseclick.eu and references therein.-   7. Chan, T. R.; Higraf, R.; Sharpless, K. B.; Fokin, V. V.    “Polytriazoles as Copper(I)—Stabilizing Ligands in Catalysis.” Org.    Lett. 2004, 6, 2853-2855.-   8. El-Sagheer, A. H.; Brown, T. “Click Chemistry with DNA.” Chem.    Soc. Rev. 2010, 39, 1388-1405.-   9. C. W. Tomoe, C. Christensen, M. Meldal, J. Org. Chem. 2002, 67,    3057-3064; V. V. Rostovtsev, L. G. Green, V. V. Fokin, K. B.    Sharpless, Angew. Chem. 2002, 114, 2708-2711; Angew. Chem. Int. Ed.    2002, 41, 2596-2599.-   10. C. J. Burrows, J. G. Muller, Chem. Rev. 1998, 98, 1109-1151.-   11. T. R. Chan, R. Hilgraf, K. B. Sharpless, V. V. Fokin, Org. Lett.    2004, 6, 2853-2855.-   12. J. Gierlich, G. A. Burley, P. M. E. Gramlich, D. M. Hammond, T.    Carell, Org. Lett. 2006, 8, 3639-3642. F. Seela, V. R. Sirivolu,    Chem. Biodiversity 2006, 3, 509-514.-   13. P. M. E. Gramlich, S. Warncke, J. Gierlich, T. Carell, Angew.    Chem. 2008, 120, 3491-3493; Angew. Chem. Int. Ed. 2008, 47,    3442-3444.

1. A compound of the formula:

wherien R¹ is LYZ; L is an optional member selected from the groupconsisting of a bond, a C₁-C₂₀ alkylene, and a C₁-C₂₀ alkenylene;wherein the alkylene or alkenylene is optionally interrupted by at leastone heteroatom; Y is an optional member selected from the groupconsisting of a bond, —O—, —S—, —NH—, —NHC(O)—, —C(O)NH—, —NR¹⁵—,—NR¹⁵C(O)—, —C(O)NR¹⁵—, —N(Z)—, —N(Z)C(O)—, and —C(O)N(Z)—; R¹⁵ is amember selected from the group consisting of alkyl andalkoxycarbonylalkyl, wherein the alkyl is optionally interrupted by atleast one heteroatom; Z is R¹⁶; or alternatively, —Y—Z is a memberselected from the group consisting of —N(Z)₂, —N(Z)C(O)Z, and—C(O)N(Z)₂, and the two Z groups may optionally be linked to form acycloalkynyl group; and R¹⁶ is member selected from the group consistingof azido, alkynyl, a pegylated azido, a pegylated alkynyl, a pegylatedcycloalkynyl, a pegylated spirocycloalkynyl, an o-diarylphosphino arylester, and an ortho substituted phosphine oxide aryl ester, DBCO,DBCO-1, TPPME, BARAC, DIFO, BCN, DIBO, TMDIBO and DIFO3.
 2. The compoundof claim 1, wherein R¹⁶ is member selected from the group consisting ofazido, alkynyl, a pegylated azido, and a pegylated alkynyl.
 3. Thecompound of claim 2, wherein R¹⁶ is azido.
 4. The compound of claim 2,wherein R¹⁶ is alkynyl.
 5. The compound of claim 1, wherein R¹⁶ ismember selected from the group consisting of DBCO, DBCO-1, TPPME, BARAC,DIFO, BCN, DIBO, TMDIBO and DIFO3.
 6. The compound of claim 5, whereinR¹⁶ is DBCO or DBCO-1.
 7. The compound of claim 5, wherein R¹⁶ is DBCO.8. The compound of claim 1, wherein said compound has the formula:


9. The compound of claim 1, wherein said compound has the formula:


10. The compound of claim 1, wherein said compound has the formula:


11. A method for imaging, said method comprising: administering acompound having the formula:

wherien R¹ is LYZ; L is an optional member selected from the groupconsisting of a bond, a C₁-C₂₀ alkylene, and a C₁-C₂₀ alkenylene;wherein the alkylene or alkenylene is optionally interrupted by at leastone heteroatom; Y is an optional member independently selected from thegroup consisting of a bond, —O—, —S—, —NH—, —NHC(O)—, —C(O)NH—, —NR¹⁵—,—NR¹⁵C(O)—, —C(O)NR¹⁵—, —N(Z)—, —N(Z)C(O)—, and —C(O)N(Z)—; R¹⁵ is amember selected from the group consisting of alkyl andalkoxycarbonylalkyl, wherein the alkyl is optionally interrupted by atleast one heteroatom; Z is -L-R¹⁶; or alternatively, —Y—Z is a memberselected from the group consisting of —N(Z)₂, —N(Z)C(O)Z, and—C(O)N(Z)₂, and the two Z groups may optionally be linked to form acycloalkynyl group; and R¹⁶ is member selected from the group consistingof azido, alkynyl, a pegylated azido, a pegylated alkynyl, a pegylatedcycloalkynyl, a pegylated spirocycloalkynyl, an o-diarylphosphino arylester, and an ortho substituted phosphine oxide aryl ester, DBCO,DBCO-1, TPPME, BARAC, DIFO, BCN, DIBO, TMDIBO and DIFO3, to a tissue ororganism.
 12. The method of imaging of claim 11, wherein said methodfurther comprises detecting said compound in a living organism.
 13. Themethod of imaging of claim 11, wherein the method comprises a step ofimaging a tumor, tissue, or organ.
 14. The method of imaging of claim13, wherein the tumor, tissue, or organ is selected from the groupconsisting of vascular endothelial tissue, an abnormal vascular wall ofa tumor, a solid tumor, a tumor of the head, a tumor of the neck, atumor of the gastrointestinal tract, a tumor of the liver, a tumor ofthe breast, a tumor of the prostate, a tumor of the ovary, a tumor ofthe uterus, a tumor of the testicle, a tumor of the lung, a nonsolidtumor, a malignant hematopoietic cell, a malignant lymphoid cell, alesion in the vascular system, a diseased bone marrow, neuronal tissue,diseased neuronal tissue, and diseased cells in which the disease is anautoimmune disease or an inflammatory disease.
 15. The method of claim11, wherein the compound is used as a detectable tracer element in abiological or non-biological fluid.
 16. The method of claim 11, whereinthe compound is used to enhance visualization of chorioretinal diseases,vascular disorders, retinopathies, neovascularization, or ocular tumors;and wherein the method comprises direct microscopic imaging.
 17. Themethod of claim 11, wherein the compound is used to enhancevisualization of skin diseases or skin tumors; and wherein the methodcomprises direct microscopic imaging.
 18. The method of claim 11,wherein the compound is used to enhance visualization of agastrointestinal disease, a gastrointestinal tumors, an oral disease, anoral tumor, a bronchial disease, a bronchial tumor, a cervical disease,cervical tumor, a urinary disease, or a urinary tumor; and wherein themethod comprises endoscopy.
 19. The method of claim 11, wherein R¹⁶ ismember selected from the group consisting of azido, alkynyl, a pegylatedazido, and a pegylated alkynyl.
 20. The method of claim 11, wherein R¹⁶is azido.
 21. The method of claim 11, wherein R¹⁶ is alkynyl.
 22. Themethod of claim 11, wherein R¹⁶ is member selected from the groupconsisting of DBCO, DBCO-1, TPPME, BARAC, DIFO, BCN, DIBO, TMDIBO andDIFO3.
 23. The method of claim 11, wherein R¹⁶ is member selected fromthe group consisting of DBCO, and DBCO-1.
 24. The method of claim 11,wherein R¹⁶ is DBCO.
 25. The method of claim 11, wherein said compoundhas the formula:


26. The method of claim 11, wherein said compound has the formula:


27. The method of claim 11, wherein said compound has the formula:


28. A method for labeling a biomolecule, said method comprising:reacting a compound of the formula:

wherien R¹ is LYZ; L is an optional member selected from the groupconsisting of a bond, a C₁-C₂₀ alkylene, and a C₁-C₂₀ alkenylene;wherein the alkylene or alkenylene is optionally interrupted by at leastone heteroatom; Y is an optional member selected from the groupconsisting of a bond, —O—, —S—, —NH—, —NHC(O)—, —C(O)NH—, —NR¹⁵—,—NR¹⁵C(O)—, —C(O)NR¹⁵—, —N(Z)—, —N(Z)C(O)—, and —C(O)N(Z)—; R¹⁵ is amember selected from the group consisting of alkyl andalkoxycarbonylalkyl, wherein the alkyl is optionally interrupted by atleast one heteroatom; Z is R¹⁶; or alternatively, —Y—Z is a memberselected from the group consisting of —N(Z)₂, —N(Z)C(O)Z, and—C(O)N(Z)₂, and the two Z groups may optionally be linked to form acycloalkynyl group; and R¹⁶ is member selected from the group consistingof azido, alkynyl, a pegylated azido, a pegylated alkynyl, a pegylatedcycloalkynyl, a pegylated spirocycloalkynyl, an o-diarylphosphino arylester, and an ortho substituted phosphine oxide aryl ester, DBCO,DBCO-1, TPPME, BARAC, DIFO, BCN, DIBO, TMDIBO and DIFO3, with abiomolecule, to label the biomolecule.